soluble versions of heterodimeric receptors, e.g., cd94/nkg2 receptors, and methods of producing and using such constructs, are described. The constructs comprise soluble fragments of, each receptor monomer, and some constructs further comprise at least one immunoglobulin Fc domain. Exemplary constructs are those wherein (1) each soluble fragment is linked to an immunoglobulin Fc domain, which are then allowed to dimerize, (2) each soluble fragment is linked to an immunoglobulin Fc domain mutated to promote forced dimerization with the correct counterpart, and (3) single-chain constructs where the monomeric receptor fragments are linked, and the C-terminal fragment is linked to an Fc domain.
|
12. A soluble cd94/NKG2A receptor fusion protein comprising the sequence of any one of SEQ ID NOS: 37-39.
1. A soluble cd94/nkg2 receptor fusion protein comprising a soluble portion of an nkg2 amino acid sequence and a soluble portion of a cd94 amino acid sequence wherein said fusion protein binds HLA-E.
2. The soluble cd94/nkg2 receptor fusion protein of
3. The soluble cd94/nkg2 receptor fusion protein of
4. The soluble cd94/nkg2 receptor fusion protein of
5. The soluble cd94/nkg2 receptor fusion protein of
6. The soluble cd94/nkg2 receptor fusion protein of
8. The soluble cd94/NKG2A receptor fusion protein of
10. The soluble cd94/NKG2C receptor fusion protein of
11. The soluble cd94/nkg2 receptor fusion protein of
14. A method of producing a soluble cd94/nkg2 receptor fusion protein, comprising culturing a cell comprising a nucleic acid encoding the soluble cd94/nkg2 receptor fusion protein of
15. A composition comprising the soluble cd94/nkg2 receptor fusion protein of
16. The soluble cd94/nkg2 receptor fusion protein of
19. A composition comprising the soluble cd94/nkg2 receptor fusion protein of
20. A method of producing a soluble cd94/nkg2 receptor fusion protein, comprising culturing a cell comprising a nucleic acid encoding the soluble cd94/nkg2 receptor fusion protein of
|
This application is a 35 U.S.C. §371 national stage application of International Patent Application PCT/EP2007/056276 (published as WO 2007/147898), filed Jun. 22, 2007, which claimed priority of European Patent Application 06115901.8, filed Jun. 22, 2006; this application further claims priority under 35 U.S.C. §119 of U.S. Provisional Application 60/817,116, filed Jun. 28, 2006.
This invention relates to soluble heterodimeric receptors for therapeutic and diagnostic applications, and to methods of designing and producing such soluble heterodimeric receptors. Exemplary compositions of the invention comprise soluble fragments of heterodimeric receptors as well as the Fc-portion of immunoglobulins.
Soluble receptors, such as soluble TNFR (Enbrel®), have proven great value for therapeutical applications. Soluble receptors may also be used for diagnostic purposes, as well, e.g., for the screening of ligand-expression on diseased tissues, such as tumor-tissues in cancer patients.
The CD94/NKG2 family of receptors is composed of heterodimeric receptor members with activating or inhibitory potential. These receptors are expressed predominantly on NK cells and a subset of CD8+T cells, and they have been shown to play an important role in regulating responses against infected and tumorigenic cells. The main ligand for the CD94/NKG2 receptor is HLA-E. U.S. Pat. No. 6,262,244 to Houchins et al. described the human NKG2A sequence (SEQ ID NO:1), and Chang et al. (Eur J Immunol. 1995;25:2433-7) reported the human CD94 sequence (SEQ ID NO:2).
Soluble versions of CD94/NKG2 receptors are of interest not only as research tools but also as therapeutic agents. Both therapeutical and diagnostic applications, however, require stable soluble receptors that can be produced efficiently in a suitable biosystem. For many heterodimeric receptors such as, e.g., CD94/NKG2, this has, so far, proven difficult. Potential reasons are homo-dimerization of the single subunits, and that the more complex structure of heterodimeric receptors makes it more difficult to design soluble versions that are sufficiently stable.
Certain soluble CD94/NKG2A constructs, based on the expression of tagged soluble portions of the CD94 and NKG2A proteins, have been proposed in the literature (Brooks et al., J Immunol 1999:162:305-13; Ding et al Scand. J. Immunol. 1999;49:459-465; and Kaiser et al. Journal of Immunology, 2005, 174: 2878-2884). Soluble versions of other multimeric receptors, some of which fused to immunoglobulin Fc portions, have been described in, e.g., WO9937772, WO200208272, and WO20023237, relating to multimeric IL-18 receptor molecules; Wu et al. (Protein Sci. 1999;8:482-9), describing soluble forms of the IL-2 receptor; WO200222153, describing a soluble IL-20 receptor; WO200212345, relating to soluble ZCY-TOR 11 cytokine receptors, WO2002101006, relating to heteromultimeric proteins such as T-cell receptors, WO9533059, describing a heterodimeric receptor of gp130 and oncostatin M receptor beta chain, and U.S. Pat. No. 6,238,890, describing soluble forms of various glycoproteins. Soluble versions of heterodimeric T-cell receptors or subunits thereof have also been described in, e.g., Clements et al., Acta Crystallogr D Biol Crystallogr. 2002;58:21314; Laugel et al., J. Biol. Chem. 2005;280:1882-1892; Kim et al., J Mol Biol. 2000;302:899-916.
Further, U.S. Patent Publication Nos. 20030195338 and 20040072256 describe various types of Fc-linked receptor sequence proteins, U.S. Pat. No. 6,018,026 relates to dimerized fusion proteins; U.S. Pat. No. 6,833,441 relates to methods for generating chimeric heteromultimers; WO92/06204 and WO03012069 relate to, e.g., techniques for producing libraries of heterodimeric receptors; U.S. Patent Publication No. 20040138417 describes heteromultimer adhesins. Also, principles to promote heteromultimer formation are described in U.S. Patent Publication No. 20030078385, and reviewed in Marvin and Zhu, Acta Pharmacol Sin 2005;26(6):649-658 (see also Kontermann, Acta Pharmacol. Sin. 2005;26:1-9.
However, there remains a need for soluble versions of heterodimeric receptors such as CD94/NKG2 for diagnostic or therapeutic applications, and efficient methods of producing stable soluble heterodimeric receptors. The present invention addresses these and other needs in the art.
The present invention provides for soluble constructs of heterodimeric receptors. The constructs comprise an essentially soluble portion of a first and second subunits of a heterodimeric receptor. In some aspects, the constructs further comprise one or two Fc-portions of an immunoglobulin molecule. In one exemplary aspect, each soluble portion is associated or covalently linked to an Fc-portion of an immunoglobulin, and the final construct comprises one of each subunit-Fc fusion or hybrid polypeptide. In another exemplary aspect, the Fc-portion of each subunit-Fc fusion or hybrid polypeptide comprises one or more mutations promoting heterodimerization of a fusion or hybrid polypeptide comprising a soluble portion of the first subunit to a fusion or hybrid polypeptide comprising a soluble portion of the second subunit, thus reducing homodimerization between identical subunit-Fc polypeptides. In yet another exemplary aspect, the soluble receptor construct is a single-chain soluble receptor-Fc fusion or hybrid protein-complex, comprising the soluble portion of the first subunit linked to the soluble portion of the second subunit. In still another exemplary aspect, the soluble receptor construct is a single-chain soluble receptor-Fc fusion or hybrid protein-complex, comprising the soluble portion of the first subunit linked to the soluble portion of the second subunit which, in turn, is linked to an Fc-portion. In one embodiment, the invention provides dimers of the single-chain receptor-Fc fusion or hybrid protein.
In one aspect, the heterodimeric receptor is an CD94/NKG2 receptor. In another aspect, the heterodimeric receptor is a CD94/NKG2A receptor.
The invention also provides for various methods of making such soluble constructs, as well as various uses of the soluble constructs according to the invention. One exemplary use is in the detection of the natural ligand (e.g., HLA-E) in a biological sample.
These and other aspects are described in more detail below and in the drawings.
The term “receptor” denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. An exemplary receptor described herein is the CD94/NKG2A receptor.
By “multimeric” or “heteromultimeric” is meant comprising two or more different subunits. A “heterodimeric” receptor contains two different subunits, herein denoted “S1” (subunit 1) and “S2” (subunit 2).
By “soluble” multimeric receptor is meant herein a multimeric receptor, each of whose subunits comprises part or all of an extracellular domain of a receptor, but lacks part or all of any transmembrane domain, and lacks all of any intracellular domain. In general, a soluble receptor of the invention is soluble in an aqueous solution. However, under certain conditions, the receptor can be in the form of an inclusion body, which is readily solubilized by standard procedures. A “soluble portion of subunit 1” can herein be denoted “sS1” whereas “a soluble portion of subunit 2” can be denoted “sS2”.
A “hybrid” protein is a protein comprising two polypeptide segments linked via at least one linkage other than a peptide bond (e.g., by chemical coupling or an affinity interaction such as via, e.g., biotin/avidin).
A “fusion” protein is a protein comprising two polypeptide segments linked by a peptide bond, produced, e.g., by recombinant processes.
The term “NKG2” includes full-length or partial polypeptides from any and all members of the NKG2 family, including, but not limited to, NKG2A, NKG2B, NKG2C, NKG2E, and NKG2F, as well a human and non-human orthologs of these members and variants thereof. Typically, the amino acid sequence of a variant of an NKG2 polypeptide is highly identical or similar to the amino acid sequence of the corresponding wild-type NKG2 polypeptide (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% identical).
The term “NKG2A” includes full-length or partial NKG2A polypeptides from both human and non-human orthologs, as well as variants thereof. Typically, the amino acid sequence of a variant of an NKG2A polypeptide is highly identical or similar to the amino acid sequence of a wild-type NKG2A polypeptide (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% identical).
The term “NKG2C” includes full-length or partial NKG2A polypeptides from both human and non-human orthologs, as well as variants thereof. Typically, the amino acid sequence of a variant of an NKG2A polypeptide is highly identical or similar to the amino acid sequence of a wild-type NKG2A polypeptide (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% identical).
The term “CD94” includes full-length or partial CD94 polypeptides from both human and non-human orthologs, as well as variants thereof. Typically, the amino acid sequence of a variant of an CD94 polypeptide is highly identical or similar to the amino acid sequence of a wild-type CD94 polypeptide (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% identical).
The term “ortholog” denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
An “Fc domain” herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and/or CH3 domains as well as fragments of such peptides comprising only, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derived from an immunoglobulin of any species and/or any subtype, including, but not limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. Exemplary Fc sequences are provided herein, e.g., in
As used herein, a “variant” polypeptide of a parent or wild-type polypeptide contains one or more amino acid substitutions, deletions and/or additions as compared to the parent or wild-type. Typically, such variants have a sequence identity to the parent or wild-type sequence of at least about 90%, at least about 95%, at least about 96%, at least about 97%, 98%, or at least about 99%, and have preserved or improved properties as compared to the parent or wild-type polypeptide. Some changes may not significantly affect the folding or activity of the protein or polypeptide; conservative amino acid substitutions, as are well known in the art, changing one amino acid to one having a side-chain with similar physicochemical properties (basic amino acid: arginine, lysine, and histidine; acidic amino acids: glutamic acid, and aspartic acid; polar amino acids: glutamine and asparagine; hydrophobic amino acids: leucine, isoleucine, valine; aromatic amino acids: phenylalanine, tryptophan, tyrosine; small amino acids: glycine, alanine, serine, threonine, methionine), small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidine tract, protein A (Nilsson et al., EMBO 1985;14:1075 et seq.; Nilsson et al., Methods Enzymol. 1991; 198:3 et seq.), glutathione S-transferase (Smith and Johnson, Gene 1988;67:31 et seq.), or other antigenic:epitope or binding domain. See, in general Ford et al., Protein Expression and Purification 1991;2:95-107. DNAs encoding affinity tags are available from commercial suppliers.
Sequence differences or “identity,” in the context of amino acid sequences, can be determined by any suitable technique, such as (and as one suitable selection in the context of this invention) by employing a Needleman-Wunsch alignment analysis (see Needleman and Wunsch, J. Mol. Biol. (1970) 48:443453), such as is provided via analysis with ALIGN 2.0 using the BLOSUM50 scoring matrix with an initial gap penalty of −12 and an extension penalty of −2 (see Myers and Miller, CABIOS (1989) 4:11-17 for discussion of the global alignment techniques incorporated in the ALIGN program). A copy of the ALIGN 2.0 program is available, e.g., through the San Diego Supercomputer (SDSC) Biology Workbench. Because Needleman-Wunsch alignment provides an overall or global identity measurement between two sequences, it should be recognized that target sequences which may be portions or subsequences of larger peptide sequences may be used in a manner analogous to complete sequences or, alternatively, local alignment values can be used to assess relationships between subsequences, as determined by, e.g., a Smith-Waterman alignment (J. Mol. Biol. (1981) 147:195-197), which can be obtained through available programs (other local alignment methods that may be suitable for analyzing identity include programs that apply heuristic local alignment algorithms such as FastA and BLAST programs). Further related methods for assessing identity are described in, e.g., International Patent Application WO 03/048185. The Gotoh algorithm, which seeks to improve upon the Needleman-Wunsch algorithm, alternatively can be used for global sequence alignments. See, e.g., Gotoh, J. Mol. Biol. 162:705-708 (1982).
The present invention provides for soluble receptor-complexes from heterodimeric receptors, all comprising a soluble portion of each of the two subunits of a heterodimeric receptor, and, in some aspects, at least one Fc-portion of an immunoglobulin molecule.
In one aspect, each soluble portion is associated or covalently linked to an Fc-portion of an immunoglobulin, and the final soluble-receptor complex is formed by associating or covalently linking the hybrid or fusion protein comprising the soluble portion of the first subunit (sS1-Fc) to the hybrid or fusion protein comprising the soluble portion of the second subunit (sS2-Fc). Linkage of the various segments may be obtained via, e.g., covalent binding such as by chemical cross-linking, peptide linkers, disulfide bridges, etc., or affinity interactions such as by avidin-biotin or leucine zipper technology.
Another aspect is similar to the one describe above, but the Fc-molecule of each fusion protein comprises specific mutations designed to produce forced heterodimerisation between the different fusion proteins, resulting in a soluble-receptor complex where fusion protein sS1-Fc1 is fused with sS2-Fc2.
In yet another aspect, the soluble receptor complex is a single-chain soluble receptor-Fc fusion or -hybrid protein-complex, comprising the soluble portion of the first subunit (sS1) linked to the soluble portion of the second subunit (sS2). In a particular embodiment, the C-terminal of one of sS1 and sS2 is further linked to an Fc-portion. Linkage of the various segments may be obtained via, e.g., covalent binding such as by chemical cross-linking, peptide linkers, disulfide bridges, etc., or affinity interactions such as by avidin-biotin or leucine zipper technology. In one embodiment, the single-chain receptor protein is a fusion protein, i.e., all linkages between different segments in the protein are via peptide linkers or via direct peptide-peptide binding.
In still another aspect, two units of the single-chain Fc-fusion protein described above are joined or linked, thus essentially forming a homodimer of a heterodimeric receptor. In one exemplary embodiment, the heterodimeric receptor is a CD94/NKG2 receptor, and the two single-chain Fc-fusion proteins are linked via a disulfide bond between cysteine residues naturally occurring in the soluble segments of the CD94 and NKG2 subunits.
The soluble receptor-complexes of heterodimeric receptors according to the invention can be more stable and thus can be stored longer. In addition, stable soluble receptor-complexes from heterodimeric receptors are suitable in applications where a longer half-life is required, e.g., for immunizations and therapeutical applications.
As described in the Examples, the N and C-terminals of CD94 and NKG2A were found to be close in an in silico CD94/NKG2A model. This confirmed the suitability of soluble CD94/NKG2 heterodimeric receptors according to the invention, since a C-terminal of a first subunit can then be linked to the N-terminal of the second subunit by a short peptide linker, and because of high sequence homology in the soluble portions of NKG2 proteins (see
The following are exemplary soluble CD94/NKG2 constructs:
1) CD94-Fc/NKG2-Fc: A stable Fc-fusion protein or hybrid-protein conjugate comprising extracellular domains of CD94 and an NKG2 protein which are both conjugated or fused to murine Fc. The protein can be produced and secreted from mammalian cells and purified by a single-step affinity-chromatography. In one exemplary embodiment, this soluble receptor complex can be illustrated by
2) CD94-Fc1/NKG2-Fc2: A stable Fc-fusion protein or -protein conjugate comprising extracellular domains of CD94 and an NKG2 protein, each of which is fused to a differently mutated murine Fc-domain. The mutations in the Fc-domain fused or conjugated to the CD94 soluble segment promote dimerization with the mutated Fc-domain fused or conjugated to the soluble NKG2 segment, and vice versa, thus avoiding CD94- or NKG2-homodimerisation. The protein can be produced and secreted from mammalian cells and purified by a single-step affinity-chromatography. In one exemplary embodiment, this soluble receptor complex can be illustrated by
3) Single-chain CD94/NKG2-Fc: A stable single-chain Fc-fusion protein comprising extracellular domains of CD94 and an NKG2 protein that are, e.g., interlinked by a serine-glycine-spacer, and/or fused or conjugated to an Fc-sequence. The protein can be produced and secreted from mammalian cells and purified by a single-step affinity-chromatography. In one exemplary embodiment, a soluble receptor Fc-fusion protein can be illustrated by
As described in Examples 18 and 19, soluble CD94/NKG2A receptors bound soluble HLA-E, as well as MAb's specific for CD94 (HP-3D9) and NKG2A (Z199), indicating that they were properly folded. The soluble CD94/NKG2A receptors tested appeared stable, since they, with few exceptions, were capable of binding soluble HLA-E tetramers, HP3D9 (Anti-CD94) and anti-NKG2A (Z199) after storage at 4° C. or −20° C. in PBS for, in some cases, several weeks. Further, as described in Examples 20 and 21, a soluble CD94/NKG2C receptors were prepared, and were found capable of binding to an anti-NKG2C-specific antibody.
The heterodimeric receptor constructs of the invention comprises soluble parts of each monomeric subunit and at least one polypeptide comprising all or part of an immunoglobulin heavy-chain constant domain (i.e., an Fc domain).
Typical heterodimeric receptors on which the present principles can be applied include, but are not limited to, the CD94/NKG2A, CD94/NKG2B, CD94/NKG2C, CD94/NKG2E, and CD94/NKG2F receptor. The NKG2 family of receptors have a high sequence homology, as shown in
CD94 (Uniprot accession No. Q13241) comprises 179 amino acids in 3 domains, a cytoplasmic region comprising residues 1-10, a transmembrane region comprising residues 11-31, and an extracellular region comprising residues 32-179, of the following sequence:
(SEQ ID NO: 2)
MAVFKTTLWRLISGTLGIICLSLMATLGILLKNSFTKLSIEPAFTPGPNI
ELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQ
NTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFETFNTK
NCIAYNPNGNALDESCEDKNRYICKQQLI.
One exemplary heterodimeric receptor is the CD94/NKG2A receptor. NKG2A (Uniprot accession No. P26715) comprises 233 amino acids in 3 domains, with a cytoplasmic domain comprising residues 1-70, a transmembrane region comprising residues 71-93, and an extracellular region comprising residues 94-233, of the following sequence:
(SEQ ID NO: 1)
MDNQGVIYSDLNLPPNPKRQQRKPKGNKSSILATEQEITYAELNLQKASQ
DFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQR
HNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSK
NSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKD
SDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL.
Another exemplary heterodimeric receptor is the CD94/NKG2C receptor. The NKG2C sequence (SEQ ID NO:3) comprises 231 amino acids in similar arrangement as described for NKG2A.
As described above, other NKG2 receptors may also be applied to construct heterodimeric receptors according to the invention. In such constructs, one monomer unit comprises a soluble segment of a CD94 sequence, and one monomer unit comprises a soluble segment of an NKG2 sequence from, e.g., human NKG2B (SEQ ID NO:70), NKG2E (SEQ ID NO:4), or NKG2F (SEQ ID NO:5).
Several non-human orthologs to NKG2A and CD94 are known that may also be used in the preparation of soluble CD94/NKG2A receptors. Non-limiting orthologues of human NKG2A include those having UNIPROT accession numbers Q95MI5 (UNIPROT-ID:NKG2A_PANTR (Chimpanzee)); Q9MZJ3 (UNIPROT-ID:NKG2A_MACMU (Rhesus macaque)); Q68VD2 (UNIPROT-ID:Q68VD2_MACFA (Cynomolgus monkey)); O54872 (UNIPROT-ID:O54872_RAT (Rat)); and Q9WU31 (UNIPROT-ID:Q9WU31_MOUSE (Mouse)). Non-limiting orthologues of human CD94 include those having UNIPROT accession numbers Q9MZ41 (UNIPROT-ID:KLRD1_PANTR (Chimpanzee)); Q8MHY9 (UNIPROT-ID:KLRD1_PONPY (Orangutan)); Q9MZK9 (UNIPROT-ID:KLRD1_MACMU (Rhesus macaque)); Q68VD4 (UNIPROT-ID:Q68VD4_MACFA (Cynomolgus monkey)); O35778 (UNIPROT-ID:O35778_RAT (Rat)); O54708 (UNIPROT-ID:O54708_MOUSE (Mouse)); and Q38HS3 (UNIPROT-ID:Q38HS3_CANFA (Dog)). These sequences are publicly available at the Uniprot website (World-wide Web address ebi.uniprot.org/index.shtml).
The immunoglobulin polypeptide of the constructs of the invention may comprise a full-length or fragment of an immunoglobulin Fc-portion, or a variant thereof. In one aspect, the immunoglobulin is a wild-type sequence of an Fc-domain, or a fragment thereof. In another aspect, the variant is designed to promote heterodimerization of the two different fusion proteins, resulting in a soluble-receptor complex where fusion protein sS1-Fc1 is fused with sS2-Fc2. In another aspect, the variant is designed to promote heterodimerization of two different single-chain single-chain receptors as described herein.
In particular aspects, the immunoglobulin heavy-chain constant domain are derived from human or non-human mammalian antibodies. In one embodiment, the constant domain is derived from a human IgG1. In another embodiment, the constant domain is derived from a human IgG4. In other aspects, the constant domain is derived from a non-human (e.g., a primate or rodent) IgG molecule (or antibody type that is recognized as being substantially similar to a human IgG in terms of composition). The phrase “derived from”, in this context refers to a polypeptide identical (100%) or highly similar in terms of amino acid sequence composition (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% identical) to a wild-type or reference (“parent”) immunoglobulin constant domain, other than any indicated changes (e.g., the below-described substitutions). The phrase “derived from” is, in this sense, not intended to indicate the method by which such an antibody or antibody fragment is generated, which may be by any suitable method, such as recombinant expression, chemical protein synthesis, etc.
In one embodiment, the portion of the Fc domain is of sufficient size and composition that it increases the in vivo half-life of the construct (e.g., due to slower clearance from the circulation) as compared to a construct lacking the Fc domain; in still another particular aspect the portion of the Fc domain is functional (i.e., imparts antibody effector function to the construct antibody)).
Exemplary wild-type immunoglobulin constant domains for use in the present constructs include SEQ ID NO:25, representing a murine Fc (mFc) sequence (clone #669: IMAGE3491766); residues Val98 to Lys324 of SEQ ID NO:21, representing a murine IgG1 sequence; residues Pro100 to Lys330 of SEQ ID NO:20, representing a human IgG1 Fc sequence; and residues Glu99 to Lys327of SEQ ID NO:24, representing a human IgG4 Fc sequence. Corresponding immunoglobulin polypeptides from other wild-type sequences (e.g., SEQ ID NOS: 12-19 and 21-23) can be made using established methods in the art.
In one aspect, the invention provides mutated versions of such wild-type immunoglobulin constant domains. It has now been discovered that pairs of amino acids in the constant domains of antibody monomers are significantly involved in the multimerization and stability of such antibody monomers (and antibody molecules as a whole in the case of antibody molecules such as IgG molecules) and can, accordingly, be modified by various methods, so as to better promote the formation of bispecific antibody monomers or molecules. Typically, such pairs of amino acids are primarily found in the heavy chains of antibody molecules (e.g., between certain amino acid residues present in the CH1 and CH3 constant regions of an IgG molecule).
For example, for human immunoglobulin G antibodies, it has been discovered that ionic forces, which contribute to cross-linking the two heavy chain (“HC”) polypeptides of the tetrameric antibody molecule, are contributed mainly by six amino acids present in the CH3 region of the antibody in the following manner: E240-K253, D282-K292, and K322-D239 (sequence position numbers refer to the amino acid starting from the beginning of CH1 (similar to UNIPROT entry IGHG1_HUMAN (SEQ ID NO:20)).
Applying this discovery to the present invention, by substituting HC amino acids of an immunoglobulin peptide conjugated, fused, or linked to a soluble segment of a first subunit of a heterodimeric receptor antigen as follows—K253E, D282K, and K322D, it is possible to significantly reduce the homodimerization or “self-pairing” of the final polypeptide (which normally occurs in the original wild-type tetrameric antibody molecule). By similarly modifying the HC sequence of a second immunoglobulin polypeptide conjugated, linked, or fused to a soluble segment of a second subunit of a heterodimeric receptor by the substitutions D239K, E240K, and K292D, homodimerization of such polypeptides is also reduced or abolished.
Co-expressing the two polypeptides, each containing soluble segment from a different heterodimeric receptor subunit, can “restore” stabilizing ionic interactions (e.g., E240-K253, D282-K292, and K322-D239) and pairing of the polypeptides, resulting in generation of a soluble heterodimeric receptor. Table 1 summarizes (in exemplary fashion) these various substitutions. Corresponding mutation sites in other exemplary constant domains can be derived by alignment (see example in
The Fc-domain of the constructs of the invention may comprise one, two or all of the mutation pairs in Table 1. Thus, in one non-limiting embodiment, a heterodimeric construct of the invention comprises a first variant Fc-domain comprising a lysine (K) at a residue corresponding to residue 239 in SEQ ID NO:20 and an aspartic acid (D) at a residue corresponding to residue 292 in SEQ ID NO:20, and a second variant Fc-domain comprising a lysine at a residue corresponding to residue 282 in SEQ ID NO:20 and an aspartic acid (D) at a residue corresponding to residue 322 in SEQ ID NO:20. This type of construct may be referred to as “double-mutation” construct herein. In another non-limiting embodiment, a heterodimeric construct of the invention comprises a first variant Fc-domain comprising lysine (K) at residues corresponding to residues 239 and 240 in SEQ ID NO:20 and an aspartic acid (D) at a residue corresponding to residue 292 in SEQ ID NO:20, and a second variant Fc-domain comprising a glutamic acid (E) at a residue corresponding to residue 253 in SEQ ID NO:20, a lysine at a residue corresponding to residue 282 in SEQ ID NO:20 and an aspartic acid (D) at a residue corresponding to residue 322 in SEQ ID NO:20. Additionally or alternatively, one or more mutation pairs may be combined with other mutation pairs. One exemplary alternative or additional mutation pair is T243Y, Y284T, referring to residue positions in SEQ ID NO: 21 (IGHG1_MOUSE).
TABLE 1
Exemplary amino acid substitution in constant domains of human
IgG1 or IgG4, referring to SEQ ID NO: 20 (UniProt IGHG1_HUMAN)
or SEQ ID NO: 24 (UniProt IGHG4_HUMAN), respectively.
Antibody 1
Antibody 2
CH3 mutations
IgG1:
IgG4:
IgG1:
IgG4:
K253E
K250E
E240K
E236K
D282K
D279K
K292D
E237K
K322D
K319D
D239K
R289D
CH1 mutations
K96E
As described herein, various wild-type or variant murine Fc domains can also be used in the heterodimeric constructs according to the invention.
In the heterodimeric receptors of the invention, different segments of a polypeptide can be linked using a variety of conventional methods. In exemplary embodiments, a soluble portion of a subunit of a heterodimeric receptor is linked to an immunoglobulin polypeptide (e.g., sS1-linker-Fc or sS2-linker-Fc), or a soluble portion of a first subunit to a soluble portion of a second subunit (e.g., sS1-linker-sS2-Fc, sS2-linker-sS1-Fc, sS1-linker1-sS2-linker2-Fc, sS2-linker1-sS1-linker2-Fc). Different segments can be linked by, e.g., (1) chemical cross-linking; (2) affinity association by appending a moiety, such as a peptide, to soluble receptor segments and/or immunoglobulin polypeptide segments, and then joining the segments via the appended moiety or moieties to form a hybrid protein; and (3) linking soluble receptor segments and/or immunoglobulin polypeptide segments to form a single polypeptide chain via a polypeptide linker, i.e., a fusion protein.
In the first linkage category, any of a variety of conventional methods can be used to chemically couple (cross-link) two polypeptide chains. Covalent binding can be achieved either by direct condensation of existing side chains (e.g., the formation of disulfide bond between cysteine residues, such as may naturally occur between the soluble portions of the CD94 and NKG2 subunits in certain CD94/NKG2 receptors) or by the incorporation of external bridging molecules. Many bivalent or polyvalent agents are useful in coupling polypeptides.
In general, the cross-linking agents used are bifunctional agents reactive, e.g., with epsilon-amino group or thiol groups. These cross-linkers can be classified into two categories: homo- and hetero-bifunctional reagents. Homobifunctional reagents can react, e.g., with free thiols (e. g., generated upon reduction of disulfide bonds), and include, e.g., 5,5′-Dithiobis-(2-nitrobenzoic acid) (DNTB), and o-phenylenedimaleimide (O-PDM), which can form a thioether bond between two polypeptides having such free thiols. Heterobifunctional reagents can introduce a reactive group onto a polypeptide that will enable it to react with a second polypeptide. For example, N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) can react with a primary amino group to introduce a free thiol group. Other chemical cross-linking agents include, e.g., carbodiimides, diisocyanates, diazobenzenes, hexamethylene diamines, dimaleimide, glutaraldehyde, 4succinimidyl-oxycarbonyl-a-methyl a(2-pyridylthio) toluene(SMPT) and N-succinimidyl-S-acetyl-thioacetate (SATA). Procedures for cross-linking polypeptides with such agents are well-known in the art. See, e.g., Pierce ImmunoTechnology Catalog & Handbook (1991) E8-E39; Karpovsky et al., J. Exp. Med. 1984;160:1686 et seq.; Liu et al. Proc. Natl. Acad. Sci. 1985;82:8648 et seq.; and U.S. Pat. No. 4,676,980.
Spacer arms between the two reactive groups of cross-linkers may have various lengths and chemical compositions. A longer spacer arm allows a better flexibility of the conjugated polypeptides. while some particular components in the bridge (e.g., a benzene group) may lend extra stability to the reactive groups or an increased resistance of the chemical link to the action of various aspects (e.g., disulfide bond resistance to reducing reagents). The use of peptide spacers such as the peptide linkers or linker peptides described below is also contemplated.
In the second category of linkage methods, conventional methods can be used to append any of a variety of moieties (e.g., peptides) to soluble receptor portions and/or immunoglobulin polypeptides, thereby generating hybrid or fusion proteins which then can be associated via the appended moieties.
In one embodiment, moieties such as biotin and avidin (streptavidin) are bound or complexed to soluble receptor portions and/or immunoglobulin polypeptides, and these moieties interact to associate the two subunits.
In another embodiment, the appended moieties are both peptides, which may herein be referred to as “dimerization-promoting peptides.” Among the wide variety of such peptide linkers that can be used are the GST (glutathione S-transferase) fusion protein, or a dimerization motif thereof; a PDZ dimerization domain; FK-506 BP (binding protein) or a dimerization motif thereof; a natural or artificial helix-turn-helix dimerization domain of p53; and Protein A or its dimerization domain, domain B. In one embodiment, the appended peptides are components of a leucine zipper. The leucine zipper moieties are often taken from the human transcription factors c-jun and c-fos.
The dimerization-promoting peptide should provide an adequate degree of flexibility to prevent the two subunits from interfering with each others' activity, for example by steric hindrance, and to allow for proper protein folding. Therefore, it may be desirable to modify a dimerization-promoting peptide by altering its length, amino acid composition, and/or conformation, e.g., by appending to it still other “secondary linker moieties” or “hinge moieties.” Among the many types of secondary linker moieties are, e.g., tracts of small, preferably neutral and either polar or nonpolar, amino acids such. as, e.g., glycine, serine, threonine or alanine, at various lengths and combinations; polylysine; or the like. Alternatively, multiples of linkers and/or secondary linker moieties can be used. It is sometimes desirable to use a flexible hinge region, such as, e.g., the hinge region of human IgG, or polyglycine repeats interrupted by serine or threonine at certain intervals.
The length and composition of a dimerization-promoting peptide can readily be selected by one of skill in the art in order to optimize the desired properties of the soluble receptor, e.g., its ability to bind to its ligand. A conventional assay for binding to antibodies is described in the Examples.
The peptides can be appended to soluble receptor portions and immunoglobulin poypeptides by a variety of methods which will be evident to one of ordinary skill in the art, e.g., chemical coupling as described above (if necessary, following derivatization of appropriate amino acid groups); attachment via biotin/avidin interactions; covalent joining of the polypeptides by art-recognized methods (e.g., using appropriate enzymes); recombinant methods; or combinations thereof.
In the third linkage category, soluble receptor portions and/or immunoglobulin polypeptides are covalently linked via a peptide linker. In this category, recombinant techniques are used to join soluble portions of each of two segments, in frame, to form a single chain polypeptide molecule. Preferably, the receptor portions are separated from one another by a linker peptide, of any length or amino acid composition, most preferably a flexible loop structure, which allows the two receptor moieties to lie at an appropriate distance from each other and in a proper aligrunent for optimal. interaction, Typical linker peptides contain tracts of small, preferably neutral and either polar or nonpolar amino acids such as, e.g., glycine, serine, threonine or alanine, at various lengths and combinations; polylysine; or the like. The peptide linker can have at least one amino acid and may have 500 or more amino acids. Preferably, the linker is less than about 100 amino acids, more preferably about 2 to 30, most preferably about 3-10 amino acids. Flexible linker domains, such as the hinge region of human IgG, or polyglycine repeats interrupted by serine or threonine at certain intervals, can be used, alone or in combination with other moieties. Exemplary linkers are those based on combinations of glycine (G), serine (S), and/or arginine (R), including, but not limited to, GS, GSS, GGSGGS (SEQ ID NO:6), and GGSGGSRSS (SEQ ID NO:7). In one embodiment, the linker linking the first and second subunits in a single-chain heterodimeric receptor is GGSGGS (SEQ ID NO:6). In another embodiment, the linker linking the first and second subunits in a single-chain heterodimeric receptor is GGSGGSRSS (SEQ ID NO:7).
Recombinant methods which can be used to generate such linear, single chain heterodimeric receptors are conventional. Furthermore, routine procedures can be used to select linker peptides and to optimize parameters so that the two soluble receptor portions are aligned at a distance and in an orientation which allow optimal function of the soluble, heterodimeric receptor. See, e.g., U.S. Pat. Nos. 4,935,233 and 4,751,180.
Of course, two subunits can be associated via any combination of the above moieties, e.g., a tandem arrangement in any relative order or orientation.
Thus, in one aspect, the present invention provides isolated nucleic acids that encode a soluble heterodimeric receptor polypeptide, with isolated nucleic acids encoding a polypeptide comprising a soluble portion of at least one monomeric receptor subunit, and at least one nucleic acid encoding an immunoglobulin peptide. In one embodiment, one nucleic acid encodes an NKG2 subunit (e.g., an NKG2A, NKG2B, NKG2C, NKG2E, or NKG2F subunit) and an immunoglobulin polypeptide, one nucleic acid encodes a CD94 subunit and an immunoglobulin polypeptide, and the heterodimeric receptor encoded binds to an antibody against the corresponding CD94/NKG2 receptor or a natural ligand of the CD94/NKG2 receptor. For example, one nucleic acid can encode an NKG2A subunit and an immunoglobulin peptide, one nucleic acid can encode a CD94 polypeptide and an immunoglobulin peptide, and the heterodimeric receptor encoded can bind to a CD94/NKG2A ligand such as HLA-E or an antibody against a CD94/NKG2A receptor, e.g., human CD94/NKG2A receptor. In another embodiment, a single isolated nucleic acid encodes a single-chain heterodimeric receptor comprising both an NKG2 subunit, a CD94 subunit, and an immunoglobulin subunit, where the heterodimeric receptor encoded binds to a CD94/NKG2 ligand such as HLA-E or an antibody against a CD94/NKG2 receptor. For example, a single isolated nucleic acid can encode a single-chain heterodimeric receptor comprising both an NKG2A (or NKG2C) subunit, a CD94 subunit, and an immunoglobulin subunit, where the heterodimeric receptor encoded binds to HLA-E or an antibody against the CD94/NKG2A (or CD94/NKG2C) receptor.
Of the above-described nucleic acids, one nucleic acid can encode a soluble portion of the NKG2A receptor set forth in SEQ ID NO:1, or a variant or ortholog thereof. In one embodiment, the nucleic acid encodes a soluble portion of the NKG2A subunit comprising residues 116-233 of SEQ ID NO:1. In another embodiment, the nucleic acid encodes a soluble portion of the NKG2A subunit comprising residues 99-233 of SEQ ID NO:1. In another embodiment, the nucleic acid encodes a fragment of the NKG2A subunit which begins with a residue selected from residues 99-116 and ends at residue 116 of SEQ ID NO.1. In another embodiment, the nucleic acid encodes a soluble portion of the NKG2A subunit consisting of residues 116-233 of SEQ ID NO:1. In another embodiment, the nucleic acid encodes a soluble portion of the NKG2A subunit consisting of residues 99-233 of SEQ ID NO:1.
In one embodiment, the nucleic acid encodes a soluble portion of the NKG2C subunit comprising residues 114-231 of SEQ ID NO:3. In another embodiment, the nucleic acid encodes a soluble portion of the NKG2C subunit comprising residues 97-231, e.g., 96-231, of SEQ ID NO:3. In another embodiment, the nucleic acid encodes a fragment of the NKG2C subunit which begins with a residue selected from residues 96-114 and ends at residue 114 of SEQ ID NO.3. In another embodiment, the nucleic acid encodes a soluble portion of the NKG2A subunit consisting of residues 114-231 of SEQ ID NO:3. In another embodiment, the nucleic acid encodes a soluble portion of the NKG2A subunit consisting of residues 96-231 of SEQ ID NO:3.One nucleic acid can also or alternatively encode a soluble portion of the CD94 receptor set forth in SEQ ID NO:2. In one embodiment, the nucleic acid encodes a soluble portion of the CD94 subunit comprising residues 58-179 of SEQ ID NO:2. In another embodiment, the nucleic acid encodes a soluble portion of the CD94 subunit comprising residues 35-179 of SEQ ID NO:2. In another embodiment, the nucleic acid encodes a fragment of the CD94 subunit which begins with a residue selected from residues 35-58 and ends at residue 179 of SEQ ID NO:2. In another embodiment, the nucleic acid encodes a soluble portion of the CD94 subunit consisting of residues 58-179 of SEQ ID NO:2. In another embodiment, the nucleic acid encodes a soluble portion of the CD94 subunit consisting of residues 35-179 of SEQ ID NO:2.
The nucleic acids of the invention may also encode additional features of the heterodimeric receptor construct, such as peptide linkers, multimerization domains, affinity tags, etc. For example, the nucleic acids may further encode one or more peptide linkers linking a soluble portion of a subunit of a heterodimeric receptor to an immunoglobulin polypeptide (e.g., sS1-linker-Fc or sS2-linker-Fc), or a soluble portion of a first subunit to a soluble portion of a second subunit (e.g., sS1-linker-sS2-Fc, sS2-linker-sS1-Fc, sS1-linker1-sS2-linker2-Fc, sS2-linker1-sS1-linker2-Fc). Exemplary linkers are those based on combinations of glycine (G), serine (S), and/or arginine (R), including, but not limited to, GS, GSS, GGSGGS (SEQ ID NO:6), and GGSGGSRSS (SEQ ID NO:7). In one embodiment, the linker linking the first and second subunits in a single-chain heterodimeric receptor is GGSGGS (SEQ ID NO:6). In another embodiment, the linker linking the first and second subunits in a single-chain heterodimeric receptor is GGSGGSRSS (SEQ ID NO:7).
In another embodiment, the nucleic acids of the expression vectors encode one or more secretory signal sequences, In one embodiment, the signal sequence is MPLLLLLPLLWAGALAMD (SEQ ID NO:30).
The nucleic acids may also encode one or more cleavage sites, particularly between different segments in a fusion protein. See, e.g., Tuan et al., Connective Tissue Research 1996;34:1-9.
In one aspect, the invention provides a nucleic acid encoding an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence listed in Table 2 with or without signal (Sig) sequences, as well as expression vectors and host cells comprising such nucleic acids. In another aspect, the invention provides variants of the amino acid sequences in Table 2. In a preferred embodiment, the encoded variant binds a natural ligand of the human CD94/NKG2A receptor, or an antibody raised against the human CD94/NKG2A receptor. In another preferred embodiment, the encoded variant binds a natural ligand of the human CD94/NKG2C receptor, or an antibody raised against the human CD94/NKG2C receptor
TABLE 2
Amino acid sequences of exemplary soluble CD94/NKG2A receptor
constructs (Sig = signal sequence; mFc = murine Fc-portion;
GS, GSS etc. = linkers based on glycine (G), serine (S),
and arginine (R)). The Fc domain nomenclature refers to the
designation in FIG. 8.
Designation/Description
SEQ ID NOS:
CD94-mFc/NKG2A heterodimer
31/32
(Sig)-(mFc)-GSS-(CD94)/(Sig)-(mFc)-GS-(NKG2A)
CD94-mFc/NKG2A heterodimer with single mutation
33/34
(Sig)-(mFc-sm-CD94)-GSS-(CD94)/(Sig)-(mFc-sm-
NKG2A)-GS-(NKG2A)
CD94-mFc/NKG2A heterodimer with double mutation
35/36
(Sig)-(mFc-dm-CD94)-GSS-(CD94)/(Sig)-(mFc-dm-
NKG2A)-GS-(NKG2A)
NKG2A-CD94 single-chain
37
hexaHis-GST NKG2A-2xGGS-CD94
NKG2A-CD94 single-chain
38
Trx-hexaHis-NKG2A-2xGGS-CD94
CD94-NKG2A-mFc single-chain
39
(Sig)-(mFc)-GS-(NKG2A)-GGS-GGS-RSS-(CD94)
CD94-NKG2C-mFc single chain
59
(Sig)-(mFc)-GS-(NKG2A)-GGS-GGS-RSS-(CD94)
In another aspect, the present invention provides a first expression vector comprising the following operably linked elements: (a) a transcription promoter; a first nucleic acid encoding a first subunit of a soluble portion of a heterodimeric receptor fused to an immunoglobulin polypeptide; and a transcription terminator; and a second expression vector comprising the following operably linked elements: (b) a second transcription promoter; a second nucleic acid encoding a second subunit of a soluble portion of a heterodimeric receptor fused to an immunoglobulin polypeptide; and a transcription terminator. In another aspect, the first and second nucleic acids are contained within a single expression vector, optionally in-frame. In another aspect, the expression vector comprises the following operably linked elements: (a) a transcription promoter; a first nucleic acid encoding a single-chain soluble heterodimeric receptor; and a transcription terminator. In one embodiment of any of the preceding aspects, the expression vector further encodes linker sequences, affinity tags, or secretory signal sequence operably linked to or comprised in the nucleic acid. In another embodiment of the preceding aspects, the heterodimeric receptor is a human CD94/NKG2 receptor. In another embodiment of the preceding aspects, the heterodimeric receptor is the human CD94/NKG2A receptor. In another embodiment of the preceding aspects, the heterodimeric receptor is the human CD94/NKG2C receptor.
In another aspect, the present invention provides a cultured cell comprising at least one of the expression vectors described above, wherein the cell expresses the polypeptides encoded by the expression vector. A single cell may contain, e.g., an expression vector encoding a single subunit of a soluble heterodimeric receptor fused to an immunoglobulin polypeptide (endoding, e.g., sS1-Fc or sS2-Fc), an expression vector encoding both first and second subunits of a soluble heterodimeric receptor, each fused to an immunoglobulin polypeptide (encoding, e.g., sS1-Fc and sS2-Fc), or an expression vector encoding a single-chain heterodimeric receptor comprising first and second subunits of a heterodimeric receptor and an immunoglobulin polypeptide (e.g., sS1-sS2-Fc or sS2-sS1-Fc).
The invention also provides methods of producing soluble heterodimeric receptors by transfecting host cells with such expression vectors, and expressing one or more nucleic acids in a cell or cell culture. In one embodiment, the cultured cell comprises an expression vector as disclosed above, wherein the first and second nucleic acids are located on the same expression vector, and the cell expresses the polypeptides encoded by the expression vector. In another embodiment, the cultured cell comprises expression vectors where the first and second nucleic acids are located on independent expression vectors and are co-transfected into the cell, and cell expresses the polypeptides encoded by the nucleic acids. In another embodiment, a first host cell is transfected with an expression vector encoding a first nucleic acid per above, and a second host cell (of the same or different type) is transfected with a nucleic acid encoding a second nucleic acid per above, and the first and second host cells separately express the polypeptides encoded by the nucleic acids. In another embodiment, the cultured cell comprises an expression vector as disclosed above, wherein the cell expresses a single-chain heterodimeric or multimeric soluble receptor polypeptide encoded by the nucleic acid. In another embodiment, the cultured cell secretes a monomeric fusion protein of a soluble portion of a heterodimeric receptor subunit and an immunoglobulin peptide. In another embodiment, the cultured cell secretes a soluble heterodimeric receptor polypeptide heterodimer or multimeric complex. In another embodiment, the cultured cell secretes a soluble CD94/NKG2 receptor that binds a CD94/NKG2 ligand or an antibody against the CD94/NKG2 receptor. In another embodiment, the cultured cell secretes a soluble CD94/NKG2A receptor that binds a CD94/NKG2A ligand or an antibody against CD94/NKG2A. In another embodiment, the cultured cell secretes a soluble CD94/NKG2C receptor that binds a CD94/NKG2C ligand or an antibody against CD94/NKG2C.
In one embodiment, the present invention provides a method of producing a soluble heterodimeric receptor polypeptide that forms a heterodimeric or multimeric complex comprising: culturing a cell as disclosed above; and isolating the soluble receptor polypeptides produced by the cell. In another embodiment, the present invention provides a method of producing a soluble heterodimeric receptor comprising: co-culturing a first cell expressing a soluble portion of a first subunit of a heterodimeric receptor linked to a first immunoglobulin polypeptide and a second cell expressing a soluble portion of a second subunit of a heterodimeric receptor linked to a second immunoglobulin polypeptide, and isolating soluble heterodimeric polypeptides formed in the culture media. In another embodiment, the present invention provides a method of producing a soluble heterodimeric receptor comprising: separately culturing a first cell expressing a soluble portion of a first subunit of a heterodimeric receptor linked to a first immunoglobulin polypeptide and a second cell expressing a soluble portion of a second subunit of a heterodimeric receptor linked to a second immunoglobulin polypeptide, separately purifying the first and second subunit fusion proteins, mixing the first and second subunit fusion proteins, and isolating soluble heterodimeric polypeptides formed. The isolating steps described above may comprise one or more purification steps according to known methods in the art.
In one aspect, the invention provides isolated soluble heterodimeric receptors comprising at least one hybrid protein, i.e., a protein where two polypeptides (typically fusion polypeptides comprising at least a receptor and an immunoglobulin segment and/or another dimerization-promoting peptide) are linked via an interaction other than a peptide bond (e.g., disulfide bonding, avidin-biotin, leucine zipper, etc.). In this method, the polypeptides are separately produced by recombinant methods, and thereafter joined.
If desired, the relative amounts of two recombinant fusion proteins produced in a single cell or cell culture can be regulated, e.g., by expressing them from promoters of different strengths. For example, if the appended peptide of a first subunit A forms homodimers at a high frequency, whereas the appended peptide of a second subunit B forms homodimers at a low frequency, one can drive the formation of the desired heterodimers by expressing much higher levels of subunit B than of A. The optimal relative amounts can be determined empirically by routine experimentation.
In another aspect, the invention provides isolated soluble heterodimeric receptors encoded by the nucleic acids described above. In one embodiment, the isolated soluble heterodimeric receptor polypeptide comprises two polypeptides, the first comprising a soluble portion of a first subunit of a heterodimeric receptor linked to an immunoglobulin polypeptide, and the second comprising a second subunit of a heterodimeric receptor linked to an immunoglobulin polypeptide. The first and second polypeptides are preferably associated, e.g., via ionic forces, covalent bonds, or a combination thereof. In soluble heterodimeric receptors comprising at least two polypeptides, the polypeptides can be associated by disulfide bonding between the receptor subunit portions or the immunoglobulin portions, and/or forced interaction via mutations in one immunoglobulin segment, as described herein. In one embodiment, one polypeptide comprises an NKG2 subunit (e.g. NKG2A, NKG2B, NKG2C, NKG2E, or NKG2F) and an immunoglobulin peptide, one polypeptide comprises a CD94 polypeptide and an immunoglobulin peptide, and the heterodimeric receptor formed binds to a CD94/NKG2 ligand such as, e.g., an antibody against the corresponding CD94/NKG2 receptor, or a natural ligand, e.g., HLA-E in the case of CD94/NKG2A, CD94/NKG2C etc. In another embodiment, the heterodimeric receptor is a single-chain polypeptide comprising soluble portions of first and second subunits of a heterodimeric receptor and an immunoglobulin polypeptide. In the case of heterodimeric NKG2 receptors, the heterodimeric receptor encoded can bind to an antibody against a CD94/NKG2 receptor, e.g., a human CD94/NKG2 receptor, or a natural ligand, e.g., HLA-E in the case of NKG2A, NKG2C etc.
The heterodimeric receptor can comprise a soluble portion of the NKG2A sequence set forth in SEQ ID NO:1, or a variant or ortholog thereof. In one embodiment, the soluble portion of the NKG2A subunit comprises residues 116-233 of SEQ ID NO:1. In another embodiment, the soluble portion of the NKG2A subunit comprises residues 99-233 of SEQ ID NO:1. In another embodiment, the soluble portion comprises a fragment of the NKG2A subunit which begins with a residue selected from residues 99-116 and ends at residue 116 of SEQ ID NO.1. In another embodiment, the soluble portion of the NKG2A subunit consists of residues 116-233 of SEQ ID NO:1. In another embodiment, the soluble portion of the NKG2A subunit consists of residues 99-233 of SEQ ID NO:1.
The heterodimeric receptor may also or alternatively comprise a soluble portion of the NKG2C sequence set forth in SEQ ID NO:3. In one embodiment, the NKG2C subunit comprises residues 114-231 of SEQ ID NO:3. In another embodiment, the soluble portion of the NKG2C subunit comprises residues 97-231, e.g., 96-231, of SEQ ID NO:3. In another embodiment, the soluble portion comprises a fragment of the NKG2C subunit which begins with a residue selected from residues 96-114 and ends at residue 114 of SEQ ID NO.3. In another embodiment, the soluble portion of the NKG2A subunit consists of residues 114-231 of SEQ ID NO:3. In another embodiment, the soluble portion of the NKG2A subunit consists of residues 96-231 of SEQ ID NO:3.
The heterodimeric receptor may also or alternatively comprise a soluble portion of NKG2B, NKG2E, comprising, e.g., the segment aligned with NKG2A-residues 99-233.
The heterodimeric receptor can comprise a soluble portion of the CD94 receptor set forth in SEQ ID NO:2, or a variant or ortholog thereof. In one embodiment, the soluble portion of the CD94 subunit comprises residues 58-179 of SEQ ID NO:2. In another embodiment, the soluble portion of the CD94 subunit comprises residues 34-179 or 35-179 of SEQ ID NO:2. In another embodiment, the soluble portion comprises a fragment of the CD94 subunit which begins with a residue selected from residues 34-58 and ends at residue 179 of SEQ ID NO:2. In another embodiment, the soluble portion of the CD94 subunit consists of residues 58-179 of SEQ ID NO:2. In another embodiment, the soluble portion of the CD94 subunit consists of residues 35-179 of SEQ ID NO:2.
In one aspect, the invention provides polypeptides having amino acid sequences at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence listed in Table 1, as well as pharmaceutical compositions comprising such polypeptides. In another aspect, the invention provides heterodimeric receptors comprising variants of the amino acid sequences in Table 2. In one embodiment, the variant binds a natural ligand of a human CD94/NKG2 receptor such as HLA-E and/or an antibody raised against a human CD94/NKG2A receptor. The variant may differ from the parent in, e.g., conservative amino acid substitutions, as described above, non-conservative amino acid substitutions, additions, and/or deletions. For example, essential amino acids in the receptor polypeptides of the present invention can be generated and identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 1989;244:1081-1085; Bass et al., Proc. Natl. Acad. Sci. USA 1991;88:4498-4502). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., ligand binding and signal transduction) to identify amino acid residues that are critical to the activity of the molecule. Sites of ligand-receptor interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 1992;255:306-312; Smith et al., J. Mol. Biol. 1992;224:899-904; Wlodaver et al., FEBS Lett. 1992;309:59-64. The identities of essential amino acids can also be inferred from analysis of homologies with related receptors.
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer Science 1988;241:53-57 or Bowie and Sauer Proc. Natl. Acad. Sci. USA 1989;86:2152-2156. Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display e.g., Lowman et al., Biochem. 1991;30:10832-10837; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204, and region directed mutagenesis (Derbyshire et al., Gene 46:145, (1986); Ner et al., DNA 7:127, (1988)). Mutagenesis methods as disclosed above can be combined with high throughput screening methods to detect activity of cloned, mutagenized receptors in host cells. Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below. Mutagenized DNA molecules that encode active heterodimeric receptors or portions thereof (e.g., ligand-binding fragments) can be recovered from the host cells and rapidly sequenced using modem equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
The following describes some additional exemplary aspects of the invention:
In one aspect, the invention provides a single-chain soluble CD94/NKG2 receptor comprising a soluble portion of an NKG2 amino acid sequence and a soluble portion of a CD94 amino acid sequence. The single-chain construct may, in one aspect, further comprise an immunoglobulin polypeptide comprising all or part of an Fc domain or variant thereof. Thus, the invention also provides a single-chain soluble CD94/NKG2 receptor comprising a soluble portion of an NKG2 amino acid sequence, a soluble portion of a CD94 amino acid sequence, and an immunoglobulin polypeptide comprising all or part of an Fc domain or variant thereof. In one embodiment, in the single-chain soluble CD94/NKG2 receptor, the C-terminal of the soluble portion of a CD94 amino acid sequence can be linked to the N-terminal of the soluble portion of an NKG2 amino acid sequence, and the C-terminal of the soluble portion of an NKG2 amino acid sequence is linked to the immunoglobulin polypeptide. In another embodiment, in the single-chain soluble CD94/NKG2 receptor, the C-terminal of the soluble portion of an NKG2 amino acid sequence is linked to the N-terminal of the soluble portion of a CD94 amino acid sequence, and the C-terminal of the soluble portion of a CD94 amino acid sequence is linked to the immunoglobulin polypeptide. In the single-chain soluble CD94/NKG2 receptor, the soluble portion of an NKG2 amino acid sequence, the soluble portion of a CD94 amino acid sequence, and, in embodiments comprising an immunoglobulin polypeptide, the immunoglobulin polypeptide, can be associated by, e.g., covalently linkage. For example, the soluble portion of an NKG2 amino acid sequence and soluble portion of a CD94 amino acid sequence can be linked by a peptide linker comprising glycine and serine.
In another aspect, the invention provides a single-chain soluble CD94/NKG2A receptor comprising a soluble portion of an NKG2A amino acid sequence and a soluble portion of a CD94 amino acid sequence. The single-chain construct may, in one aspect, further comprise an immunoglobulin polypeptide comprising all or part of an Fc domain or variant thereof. Thus, the invention also provides a single-chain soluble CD94/NKG2A receptor comprising a soluble portion of an NKG2A amino acid sequence, a soluble portion of a CD94 amino acid sequence, and an immunoglobulin polypeptide comprising all or part of an Fc domain or variant thereof. In one embodiment, in the single-chain soluble CD94/NKG2A receptor, the C-terminal of the soluble portion of a CD94 amino acid sequence can be linked to the N-terminal of the soluble portion of an NKG2A amino acid sequence, and the C-terminal of the soluble portion of an NKG2A amino acid sequence is linked to the immunoglobulin polypeptide. In another embodiment, in the single-chain soluble CD94/NKG2A receptor, the C-terminal of the soluble portion of an NKG2A amino acid sequence is linked to the N-terminal of the soluble portion of a CD94 amino acid sequence, and the C-terminal of the soluble portion of a CD94 amino acid sequence is linked to the immunoglobulin polypeptide. In the single-chain soluble CD94/NKG2A receptor, the soluble portion of an NKG2A amino acid sequence, the soluble portion of a CD94 amino acid sequence, and, in embodiments comprising an immunoglobulin polypeptide, the immunoglobulin polypeptide, can be associated by, e.g., covalently linkage. For example, the soluble portion of an NKG2A amino acid sequence and soluble portion of a CD94 amino acid sequence can be linked by a peptide linker comprising glycine and serine.
In another aspect, the invention provides a single-chain soluble CD94/NKG2C receptor comprising a soluble portion of an NKG2C amino acid sequence and a soluble portion of a CD94 amino acid sequence. The single-chain construct may, in one aspect, further comprise an immunoglobulin polypeptide comprising all or part of an Fc domain or variant thereof. Thus, the invention also provides a single-chain soluble CD94/NKG2C receptor comprising a soluble portion of an NKG2C amino acid sequence, a soluble portion of a CD94 amino acid sequence, and an immunoglobulin polypeptide comprising all or part of an Fc domain or variant thereof. In one embodiment, in the single-chain soluble CD94/NKG2C receptor, the C-terminal of the soluble portion of a CD94 amino acid sequence can be linked to the N-terminal of the soluble portion of an NKG2C amino acid sequence, and the C-terminal of the soluble portion of an NKG2C amino acid sequence is linked to the immunoglobulin polypeptide. In another embodiment, in the single-chain soluble CD94/NKG2C receptor, the C-terminal of the soluble portion of an NKG2C amino acid sequence is linked to the N-terminal of the soluble portion of a CD94 amino acid sequence, and the C-terminal of the soluble portion of a CD94 amino acid sequence is linked to the immunoglobulin polypeptide. In the single-chain soluble CD94/NKG2C receptor, the soluble portion of an NKG2C amino acid sequence, the soluble portion of a CD94 amino acid sequence, and, in embodiments comprising an immunoglobulin polypeptide, the immunoglobulin polypeptide, can be associated by, e.g., covalently linkage. For example, the soluble portion of an NKG2C amino acid sequence and soluble portion of a CD94 amino acid sequence can be linked by a peptide linker comprising glycine and serine.
In another aspect, the invention provides a dimer of any single-chain soluble CD94/NKG2, CD94/NKG2A or CD94/NKG2C receptor described above.
In another aspect, the invention provides a soluble CD94/NKG2 receptor comprising an NKG2 subunit comprising a soluble portion of an NKG2 amino acid sequence and a CD94 subunit comprising a soluble portion of an CD94 amino acid sequence, wherein at least one of the NKG2 subunit and CD94 subunit comprises an immunoglobulin polypeptide comprising all or part of an Fc domain or variant thereof, wherein non-limiting examples of NKG2 subunits are NKG2A and NKG2C. The immunoglobulin polypeptide may, for example, comprise a portion of an IgG Fc domain which increases the in vivo half-life of the construct. In one embodiment, the immunoglobulin polypeptide is a functional Fc domain. In another embodiment, the Fc domain is from an IgG4 antibody. In yet another embodiment, the Fc domain is from an IgG1 antibody.
In another aspect, only one of the NKG2 and CD94 subunits is linked to an immunoglobulin polypeptide. The immunoglobulin polypeptide can, for example, be linked to the C-terminal portion of the subunit. In one embodiment, the NKG2 and CD94 subunits are linked via a peptide linker. For example, the peptide linker may comprise the sequence GGSGGS (SEQ ID NO:6) or GGSGGSRSS (SEQ ID NO:7). In a particular embodiment, the NKG2A subunit is covalently bound to the immunoglobulin polypeptide. In an alternative embodiment, the CD94 subunit is covalently bound to the immunoglobulin polypeptide.
In another aspect, each of the NKG2 and CD94 subunits is covalently bound to an immunoglobulin polypeptide. For example, the N-terminal of the NKG2 subunit can be linked to the C-terminal of a first immunoglobulin polypeptide so as to form a first polypeptide, and the N-terminal of the CD94 subunit can be linked to the C-terminal of a second immunoglobulin polypeptide so as to form a second polypeptide. In one embodiment, both the first and second immunoglobulin polypeptides are variants of a human IgG1 Fc domain, the first immunolobulin polypeptide comprising substitutions corresponding to K253E, D282K, and K322D, and the second immunoglobulin polypeptide comprising substitutions corresponding to D239K, E240K, and K292D, or vice versa. In another embodiment, both the first and second immunoglobulin polypeptides are variants of a human IgG4 Fc domain, the first immunolobulin polypeptide comprising substitutions corresponding to K250E, D279K, and K319D, and the second immunoglobulin polypeptide comprising substitutions corresponding to E236K, E237K, R289D, or vice versa.
In any of the preceding aspects, the NKG2 subunit can be NKG2A wherein the NKG2A amino acid sequence can be, for example, that of human NKG2A (SEQ ID NO:1). For example, the NKG2A subunit may comprise residues 99-233 of SEQ ID NO: 1.
In any of the preceding aspects, the NKG2 subunit can alternatively be NKG2C wherein the NKG2C amino acid sequence can be, for example, that of human NKG2C (SEQ ID NO:3). For example, the NKG2C subunit may comprise residues 96-231 of SEQ ID NO:3.
In any of the preceding aspects, the CD94 amino acid sequence can be SEQ ID NO:2. For example, the CD94 subunit may comprise residues 35-179 of SEQ ID NO:2.
In any of the preceding aspects, the immunoglobulin polypeptide may comprise full-length or fragment of a sequence selected from SEQ ID NOS:12-29. A heterodimeric construct comprising a full-length or fragment of each of SEQ ID NOS:12-29 is a specific and separate embodiment according to the invention.
In any of the preceding aspects, the immunoglobulin polypeptide can be covalently bound to a signal sequence. For example, the signal sequence may comprise the sequence of SEQ ID NO:30.
In additional or alternative aspects, the invention provides soluble CD94/NKG2A receptor comprising the sequences of SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NO:37; SEQ ID NO:38, and SEQ ID NO:39 or a soluble CD94/NKG2C receptor comprising SEQ ID NO:59.
In another aspect, the invention provides nucleic acids encoding the soluble CD94/NKG2 receptor of any of the preceding aspects. Also provided for is a cell transformed with an expression vector comprising such nucleic acids, where the cell may be, e.g., a prokaryotic cell or eukaryotic cell. Also provided for is a method of producing a soluble CD94/NKG2 receptor, comprising culturing such a cell under conditions suitable for expression of the soluble CD94/NKG2 receptor.
In another aspect, the invention provides for a pharmaceutical composition comprising an effective amount of the soluble CD94/NKG2 receptor of any of the preceding aspects, and a pharmaceutically acceptable carrier or excipient.
In another aspect, the invention provides for a method of producing an antibody against a CD94/NKG2 receptor such as, e.g., CD94/NKG2A, the method comprising: inoculating an animal with a soluble CD94/NKG2 receptor of any of the preceding aspects, wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
Preparation of Soluble Heterodimeric Receptors
The heterodimeric receptor polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells is disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989), which is incorporated herein by reference.
In general, a DNA sequence encoding a soluble heterodimeric receptor polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of, replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. Multiple components of a soluble receptor complex can be co-transfected on individual expression vectors or be contained in a single expression vector. Such techniques of expressing multiple components of protein complexes are well known in the art.
To direct a heterodimeric receptor polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) can be provided in the expression vector. The secretory signal sequence may be that of the receptor, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is joined to the heterodimeric receptor polypeptide DNA sequence in the correct reading frame. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
Cultured mammalian cells are preferred hosts within the present invention.
Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 1 1978;4:725 et seq.; Corsaro and Pearson, Somatic Cell Genetics 1981;7:603 et seq.: Graham and Van der Eb, Virology 1973;52:456 et seq., electroporation (Neumann et al., EMBO J. 1982;1:841-845), DEAE dextran mediated transfection (Ausubel et al., eds., Current Protocols in Molecular Biology, (John Wiley and Sons, Inc., N.Y., 1987), and liposome-mediated transfection (Hawley-Nelson et al., Focus 1993;15:73 et seq.; Ciccarone et al., Focus 1993;15:80 et seq.), which publications are incorporated herein by reference. The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Pahniter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314),293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. In one embodiment, the cell is dependent upon an exogenously supplied hematopoietic growth factor for its proliferation. Preferred cell lines of this type are the human TF-1 cell line (ATCC number CRL-2003) and the AML-193 cell line (ATCC number CRL-9589), which are GM-CSF-dependent human leukemic cell lines and BaF3 (Palacios and Steinmetz, Cell 41: 727-734, (1995)) which is an IL-3 dependent murine pre-B cell line. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters are preferred, such as promoters from SV40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4, 956,288. Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4, 579,821 and 4,601,978) and the adenovirus major late promoter.
Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants”. Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.” A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neornycin-type drug, such as G418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as “amplification.”
Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.
Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No. 4,775,624; and WIPO publication WO 94/06463, which are incorporated herein by reference. The use of Agrobacteriuin rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, (1987).
Fungal cells, including yeast cells, and particularly cells of the genus Saccharomyces, can also be used within the present invention, such as for producing fusion polypeptides. Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977, 092) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces ponibe, Kluyveroinyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillennondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 1986;132:3459-3465 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for transforming Acreinonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed by Lainbowitz, U.S. Pat. No. 4,486,533.
Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell.
Preferably, a soluble heterodimeric receptor of the invention is “isolated,” e.g., is in a form other than it occurs in nature, for example in a buffer, in a dry form awaiting reconstitution, as part of a kit or a pharmaceutical composition, etc. The term “isolated polypeptide” refers to a soluble heterodimeric receptor of the present invention that (1) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates or other materials (i.e., contaminants) with which it is naturally associated. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment which would interfere with its therapeutic, diagnostic, prophylactic or research use.
A variety of conventional methods can be used to isolate and/or purify a soluble heterodimeric receptor of the invention, or to isolate and/or purify its different polypeptide components prior to joining them. Soluble receptors of the invention and/or their subunits can be recovered from cells either as soluble proteins (preferably after having been secreted into the culture fluid) or as inclusion bodies, from which they may be extracted quantitatively, e.g., by 8M guanidium hydrochloride and dialysis. Conventional purification methods which can be used include, e.g., ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, and/or -gel filtration. In a preferred embodiment, affinity chromatography is used, e.g., with a column containing protein A or protein G, which can bind to the Fc moieties present in certain soluble receptors. In another embodiment, each polypeptide is “tagged” with a moiety, preferably a cleavable one, that can bind to an appropriate affinity column. For example, one or both subunits can be tagged with poly His (e.g., H′S6) to allow rapid purification by metal-chelate chromatography; with a Strep-tag which binds to streptavidin and can be cluted with iminobiotin; with maltose binding protein (MBP), which binds to ainylose and can be eluted with maltose; or with any other such moiety which can be separated by affinity chromatography. Alternatively, one can tag one or both of the subunits with epitopes to which antibodies are available, such as the FLAGO peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Conn.). For typical methods of using affinity tags, see, e.g., Recombinant Protein Protocols: Detection and Isolation, Edited by Rocky S. Tuan, Methods in Molecular Biology, Vol. 63, Humana Press, 1997 and Examples 5 and 6.
Combinations of any of the above types of purification methods can also be used.
The purity of the receptors can be determined using standard methods including, e.g., polyacrylamide gel electrophoresis, column chromatography, and amino-terminal amino acid sequence analysis.
Binding of the soluble heterodimeric receptors to, e.g., a natural ligand of the heterodimeric receptor or an antibody against the native heterodimeric receptor can be by assessed by conventional methods. A preferred assay system employing a ligand-binding receptor construct uses a commercially available biosensor instrument (BIAcoreTm, Pharmacia Biosensor, Piscataway, N.J.), wherein the ligand, an antibody, or a fragment thereof is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-240, (1991) and Cunningham and Wells, J. Mol. Biol. 234:554-563, (1993). The ligand, antibody, or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If the soluble heterodimeric receptor construct in the sample binds the ligand or antibody, it will bind to the immobilized ligand or antibody, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
Ligand-binding receptor polypeptides can also be evaluated by other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity. See, Scatchard, Ann. NY Acad. Sci. 1949;51: 660-672, and calorimetric assays (Cunningham et al., Science 1991;253:545-548; Cunningham et al., Science 1991;254:821-825).
The stability of the receptor can be tested by storing it under different conditions (e.g., temperature, pH, in different buffers) for various lengths of time. Functional stability is then tested by analyzing the binding capacity. For example, soluble CD94/NKG2A or CD94/NKG2C can be tested for its capacity to bind its ligand (HLA-E) or specific anti-CD94/NKG2A or CD94/NKG2C antibodies (e.g. Z199, Z270, HP-3D9, FAB138P) in a BiaCore or similar assay after storage for various periods of time and under different storage conditions. The percentage of binding of a stored CD94/NKG2 in comparison with a reference soluble CD94/NKG2 (which may, e.g., have been stored under standard conditions) can be a measure of its stability.
Use Soluble Heterodimeric Receptors
Particular aspects of the invention relate to the use of the soluble heterodimeric receptor constructs as immunization agents, research and selection tools, diagnostic reagents, and therapeutic agents.
In one aspect, the invention provides a method for immunizing an animal with a soluble heterodimeric receptor according to the invention in order to elicit an antibody response against the heterodimeric receptor. Exemplary advantages of using the soluble heterodimeric receptor constructs of the invention instead of soluble fragments alone include longer half-life, correct three dimensional folding of the receptor and an efficient take-up in APC's facilitated by the Fc tag (thus promoting a more robust antibody response. Antibodies against the heterodimeric receptor can then be harvested from blood (polyclonal antibody preparation) or prepared from antibody-producing B cells using hybridoma techniques or other establish technologies for monoclonal antibody production. In one embodiment, the present invention provides a method of producing an antibody to a soluble heterodimeric receptor comprising: immunizing an animal with a soluble heterodimeric receptor described herein, wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
In another aspect, the present invention provides an antibody produced by the method as disclosed above, which specifically binds to a soluble heterodimeric or multimeric receptor complex comprising a soluble heterodimeric receptor as described herein. In one embodiment, the antibody disclosed above is a monoclonal antibody.
The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)). The soluble heterodimeric receptor, or a fragment thereof, is suspended or dissolved in a buffer, optionally with an adjuvant, such as complete Freund's adjuvant. Methods for determining the amount of immunogen, types of buffers and amounts of adjuvant are well known to those of skill in the art. These parameters may be different for different immunogens, but are easily elucidated.
Similarly, the location and frequency of immunization sufficient to stimulate the production of antibodies are also well known in the art. In a typical immunization protocol, the non-human animals are injected intraperitoneally with antigen on day 1 and again about a week later. This is followed by recall injections of the antigen around day 20, optionally with adjuvant such as incomplete Freund's adjuvant. The recall injections are performed intravenously and may be repeated for several consecutive days. This is followed by a booster injection at day 40, either intravenously or intraperitoneally, typically without adjuvant. This protocol results in the production of antigen-specific antibody-producing B cells after about 40 days. Other protocols may also be utilized as long as they result in the production of B cells expressing an antibody directed to the antigen used in immunization.
For polyclonal antibody preparation, serum is obtained from an immunized non-human animal and the antibodies present therein isolated by well-known techniques. The serum may be affinity purified using any of the immunogens set forth above linked to a solid support so as to obtain antibodies that react with the soluble heterodimeric receptor or cells expressing the native heterodimeric receptor.
For monoclonal antibodies production, a non-human mammal is immunized with any soluble heterodimeric receptor described herein, including any full-length sequence, any extracellular sequence, or any fragment thereof. Splenocytes are then isolated and subsequently fused with an immortalized cell to form an antibody-producing hybridoma. The isolation of splenocytes from a non-human mammal is well-known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces and squeezing the splenocytes from the splenic capsule and through a nylon mesh of a cell strainer into an appropriate buffer so as to produce a single cell suspension. The cells are washed, centrifuged and resuspended in a buffer that lyses any red blood cells. The solution is again centrifuged and remaining lymphocytes in the pellet are finally resuspended in fresh buffer.
Once isolated and present in single cell suspension, the lymphocytes are fused to an immortal cell line. This is typically a mouse myeloma cell line, although many other immortal cell lines useful for creating hybridomas are known in the art. Preferred murine myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. U.S.A., or X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Md. U.S.A. The fusion is effected using polyethylene glycol or the like. The resulting hybridomas are then grown in selective media that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
The hybridomas are typically grown on a feeder layer of macrophages. The macrophages are preferably from littermates of the non-human mammal used to isolate splenocytes and are typically primed with incomplete Freund's adjuvant or the like several days before plating the hybridomas. Fusion methods are described in (Goding, “Monoclonal Antibodies: Principles and Practice,” pp. 59-103 (Academic Press, 1986)).
The cells are allowed to grow in the selection media for sufficient time for colony formation and antibody production; usually between 7 and 14 days. The hybridoma colonies are then assayed for the production of antibodies that bind the soluble heterodimeric receptor or cells expressing the native heterodimeric receptor. The assay is typically a calorimetric ELISA-type assay, although several other types of assays may be employed, including immunoprecipitation, radioimmunoassay, Biacore assays (see above), or Scintillation Proximity assays (SPA), as well known in the art. The wells positive for the desired antibody production are examined to determine if one or more distinct colonies are present. If more than one colony is present, the cells may be recloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody. Positive wells with a single apparent colony are typically re-cloned and re-assayed to insure only one monoclonal antibody is being detected and produced.
Hybridomas that are confirmed to be producing a monoclonal antibody of this invention are then grown up in larger amounts in an appropriate medium, such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can be grown in vivo as ascites tumors in an animal.
After sufficient growth to produce the desired monoclonal antibody, the growth media containing monoclonal antibody (or the ascites fluid) is separated away from the cells and the monoclonal antibody present therein is purified. Purification is typically achieved by chromatography using protein A or protein G-Sepharose, or an anti-mouse 1 g linked to a solid support such as agarose or Sepharose beads (all described, for example, in the Antibody Purification Handbook, Amersham Biosciences, publication No. 18-1037-46, Edition AC, the disclosure of which is hereby incorporated by reference). The bound antibody is typically eluted from protein A/protein G columns by using low pH buffers (glycine or acetate buffers of pH 3.0 or less) with immediate neutralization of antibody-containing fractions. These fractions are pooled, dialyzed, and concentrated as needed.
Alternatively, the DNA encoding an antibody of the invention is isolated from the hybridoma, placed in an appropriate expression vector for transfection into an appropriate host. The host is then used for the recombinant production of the antibody, or variants thereof, such as a humanized version of that monoclonal antibody, active fragments of the antibody or chimeric antibodies comprising the antigen-recognition portion of the antibody.
In an additional or alternative aspect of the invention, the soluble receptors of the invention such as e.g., soluble CD94/NKG2 receptors, can be used in methods for selecting NKG2-binding and/or CD94-binding agents for therapeutic or diagnostic applications. For example, one or more single-chain soluble CD94/NKG2 receptors may be used in such methods, such as, e.g., single-chain CD94/NKG2A, single-chain CD94/NKG2C, single-chain CD94/NKG2B, single-chain CD94/NKG2E and/or single-chain CD94/NKG2F. Exemplary agents include, but are not limited to, antibodies against CD94, NKG2A, NKG2B, NKG2C, NKG2E, and/or NKG2F as well as antigen-binding fragments or derivatives of such antibodies. For exemplary antibody fragments, see, e.g., Holliger and Hudson (Nat Biotechnol 2005;23:1126-1136).
In one embodiment, the invention thus provides for a method of producing an anti-NKG2 antigen-binding compound, comprising: (a) providing an antigen-binding compound that specifically binds to an NKG2 polypeptide; (b) testing the antigen-binding compound for binding to a soluble CD94/NKG2A receptor described herein; (c) selecting the antigen-binding compound if it is determined that the antigen-binding compound binds to the soluble CD94/NKG2A receptor; and (d) optionally, producing a quantity of the selected antigen-binding compound.
In another embodiment, the invention provides for a method of producing an anti-NKG2 antigen-binding compound, the method comprising: (a) producing a quantity of an antigen-binding compound that specifically binds to an NKG2 polypeptide; (b) testing a sample from said quantity of an antigen-binding compound for binding to a soluble CD94/NKG2 receptor described herein; (c) selecting the quantity for use as a medicament and/or in the manufacture of a medicament if it is determined that the antigen-binding compound binds to the soluble CD94/NKG2 receptor; and (d) optionally, preparing the quantity for administration to a human, optionally formulating a quantity of the selected antigen-binding compound with a pharmaceutically acceptable carrier.
In another embodiment, the invention provides for a method of producing an anti-NKG2 antigen-binding compound, the method comprising: (a) providing a plurality of antigen-binding compounds that specifically bind to an NKG2 polypeptide, (b) testing each of the antigen-binding compounds for binding to a soluble CD94/NKG2 receptor described herein; (c) selecting an antigen-binding compound if it is determined that the antigen-binding compound binds to said soluble CD94/NKG2 receptor; and (d) optionally, making the antigen-binding compound suitable for human administration; and/or (e) optionally, producing a quantity of the selected antigen-binding compound.
The above methods may also comprise making the antigen-binding compound human-suitable by, e.g., making the antigen-binding compound chimeric or humanized. The above methods may also comprise producing a quantity of antigen-binding compound comprises culturing a cell expressing the antigen-binding compound in a suitable medium and recovering the antigen-binding compound. In specific embodiments, the antigen-binding compound in the above methods may comprise an antibody or an antigen-binding fragment thereof. The NKG2 polypeptide may be selected from, e.g., NKG2A, NKG2B, NKG2C, NKG2E, and/or NKG2F. For example, the antigen-binding compound may bind to one or more of NKG2A, NKG2B, NKG2C, NKG2E, and NKG2F. In a specific embodiment, the anti-gen-binding compound binds at least NKG2A. In another specific embodiment, the antigen-binding compound binds at least NKG2C.
The invention also relates to methods of detecting HLA-E molecules (in, e.g., experimental or diagnostic assays), comprising contacting a sample which may contain HLA-E molecules with a soluble heterodimeric receptor according to the invention. In one embodiment, the soluble heterodimeric receptor is a single-chain CD94/NKG2A or CD94/NKG2C receptor construct as described herein. The soluble heterodimeric receptor may be labeled with a conventional detectable moiety used for such purposes, e.g., a radioactive or fluorescent entity. Alternatively, indirect methods such as, e.g., conjugating the soluble heterodimeric receptor to an enzyme capable of converting a non-detectable substrate to a detectable product, or a secondary antibody against an Fc-portion of the soluble heterodimeric receptor, may be used. Such assays can, of course, be quantitative. In one embodiment, such an assay is used to determine whether a biological sample taken from a patient contains cells that express HLA-E at the cell surface. In another embodiment, such an assay is used to determine whether an agent of interest causes an increase or decrease in a cell of the amount of HLA-E which is available for binding to CD94/NKG2A or CD94/NKG2C (e.g., human or murine cells; in a test tube, tissue sample, in culture, or in an animal) and/or whether it modulates (inhibits or enhances) the biological activity of HLA-E (e.g., its binding to a soluble receptor). Exemplary biological samples include, but are not limited to, tumor samples obtained by, e.g., biopsy or surgery; and blood samples.
In another aspect, the invention provides a method for specifically targeting HLA-E expressing or HLA-E over-expressing cells in a subject by administering a soluble heterodimeric receptor according to the invention. The Fc-portion of the soluble CD94/NKG2A or CD94/NKG2C can then function as effector domain to induce cytolysis of the HLA-E expressing cell via a CDC and/or an ADCC response.
In another aspect, a soluble heterodimeric receptor is used for purification of a receptor ligand or an antibody against the native receptor. The soluble heterodimeric receptor is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. The soluble heterodimeric receptor may also be attached via its Fc-portion to, e.g., a Protein A or Protein G affinity column. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting media can generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor. The ligand is then eluted using changes in salt concentration or pH to disrupt ligand-receptor binding.
Cytotoxicity Assays
In a particular embodiment, the invention provides agents such as soluble CD94/NKG2A receptor constructs and antibodies generated against such soluble CD94/NKG2A receptor constructs. To determine whether such constructs or antibodies can reduce or block CD94/NKG2A interactions with HLA-E, and thereby increases the cytotoxicity of CD94/NKG2A-restricted lymphocytes, the following tests can be performed.
NK cell activity can be assessed using a cell based cytotoxicity assays, e.g., measuring chromium release or other parameter to assess the ability of the antibody to stimulate NK cells to kill target cells such as P815, K562 cells, or appropriate tumor cells as disclosed in Sivori et al., J. Exp. Med. 1997;186:1129-1136; Vitale et al., J. Exp. Med. 1998; 187:2065-2072; Pessino et al. J. Exp. Med. 1998;188:953-960; Neri et al. Clin. Diag. Lab. Immun. 2001;8:1131-1135; Pende et al. J. Exp. Med. 1999;190:1505-1516, the entire disclosures of each of which are herein incorporated by reference. In one exemplary assay, the ability of an agent to reduce CD94/NKG2A-mediated signalling can be tested in a standard 4-hour in vitro cytotoxicity assay using, e.g., NKL cells that express CD94/NKG2A, and target cells that express HLA-E. Such NKL cells do not efficiently kill targets that express HLA-E because CD94/NKG2A recognizes HLA-E, leading to initiation and propagation of inhibitory signalling that prevents lymphocyte-mediated cytolysis. Such an in vitro cytotoxicity assay can be carried out by standard methods that are well known in the art, as described for example in Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993). The target cells are labeled with 51Cr prior to addition of NKL cells, and then the killing is estimated as proportional to the release of 51Cr from the cells to the medium, as a result of killing. Addition of an agent that prevents CD94/NKG2A from binding to HLA-E results in prevention of the initiation and propagation of inhibitory signalling via CD94/NKG2A. Therefore, addition of such agents results in increases in lymphocyte-mediated killing of the target cells. This step thereby identifies agents that prevent CD94/NKG2A-induced negative signalling by, e.g., blocking ligand binding. In a particular 51Cr-release cytotoxicity assay, CD94/NKG2A-expressing NKL effector-cells can kill HLA-E-negative LCL 721.221 target cells, but less efficient HLA-E-expressing LCL 721.221-Cw3 cells. In contrast, YTS effector-cells that lack CD94/NKG2A kill both cell-lines efficiently. Thus, NKL effector cells cannot kill HLA-E+ LCL 721.221-Cw3 cells due to HLA-E-induced inhibitory signalling via CD94/NKG2A. When NKL cells are pre-incubated with blocking anti-CD94/NKG2A antibodies according to the present invention in such a 51Cr-release cytotoxicity assay, HLA-E-expressing LCL 721.221-Cw3 cells are killed in an antibody-concentration-dependent fashion.
The inhibitory or potentiating activity of a receptor or antibody composition of this invention can be assessed in any of a number of other ways, e.g., by its effect on intracellular free calcium as described, e.g., in Sivori et al., J. Exp. Med. 1997;186:1129-1136, the disclosure of which is herein incorporated by reference.
NK cell activity can also be addressed using a cytokine-release assay, wherein NK cells are incubated with the receptor construct or antibody to stimulate the cytokine production of the NK cells (for example IFN-γ and TNF-α production). In an exemplary protocol, IFN-γ production from PBMC is assessed by cell surface and intracytoplasmic staining and analysis by flow cytometry after 4 days in culture. Briefly, Brefeldin A (Sigma Aldrich) is added at a final concentration of 5 μg/ml for the last 4 hours of culture. The cells are then incubated with anti-CD3 and anti-CD56 mAb prior to permeabilization (IntraPrep™; Beckman Coulter) and staining with PE-anti-IFN-γ or PE-IgG1 (Pharmingen). GM-CSF and IFN-γ production from polyclonal activated NK cells are measured in supernatants using ELISA (GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, Minn, IFN-: OptEIA set, Pharmingen).
The compounds of this invention, including receptor constructs and antibodies, may exhibit partial inhibitory activity, e.g., partially reduce the CD94/NKG2A-mediated inhibition of NK cell cytotoxicity. In one embodiment, an antibody preparation causes at least a 10% augmentation in NK cytotoxicity, preferably at least a 40% or 50% augmentation in NK cytotoxicity, or more preferably at least a 70% augmentation in NK cytotoxicity. Most preferred compounds are able to inhibit (or stimulate, in the case of activating receptors) at least 20%, preferably at least 30%, 40% or 50% or more of the activity of the NK cell, e.g. as measured in a cell toxicity assay, in comparison to cells in the absence of the compound. Also preferred, the compound can provide an increase of depletion of target cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000%, or more relative to the depletion level in the absence of the compound. Alternatively, the compounds of this invention are able to induce the lysis of matched or HLA compatible or autologous target cell population, i.e., cell population that would not be effectively lysed by NK cells in the absence of said antibody. Accordingly, compounds of this invention may also be defined as facilitating NK cell activity in vivo.
Pharmaceutical Compositions
The invention also provides compositions that comprise a soluble heterodimeric receptor or antibody (including fragments and derivatives thereof), in any suitable vehicle in an amount effective to detectably potentiate lymphocyte cytotoxicity in a patient or in a biological sample comprising lymphocytes and, optionally, HLA-E-expressing target cells. The composition further comprises a pharmaceutically acceptable carrier or excipient. Such carriers and excipients are well-known in the art, and are described in, e.g., Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
The term “biological sample” as used herein includes but is not limited to a biological fluid (for example serum, lymph, or blood), cell sample or tissue sample (for example bone marrow).
Further aspects and advantages of this invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application.
This Example describes in silico modelling identifying CD94/NKG2A segments covering the interaction with HLA-E. The protein data base (PDB; Protein Data Bank) referred to herein is described in: H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne: The Protein Data Bank. Nucleic Acids Research, 28 pp. 235-242 (2000). PDB identifiers are always 4 alphanumeric characters, e.g., 1NKR & 1OM3, sometimes referred to as 1 NKR.pdb & 1OM3.pdb.
A model of CD94/NKG2A complexed with its ligand HLA-E was built in silico. For this, a model of the CD94/NKG2A heterodimer was built using the X-ray structure of the CD94 homodimer as a homology model (PDB-ID:1B6E). In addition, the resolved crystal structure of HLA-E was used (PDB-ID:1MHE). The complex of CD94/NKG2A and HLA-E was then built in silico, using the X-ray structure of the related NKG2D-MICA complex (PDB-ID:1HYR). The complete model is shown in
The model was consistent with mutational studies which identify the HLA-E residues R65, D69, Q72, R75, R79, H155, D162, E166 as the interacting residues (H. Wada et al., Eur. J. Immunol. 2004. 34, 81-90). In the alignment of CD94, NKG2A and NKG2D, the model also identifies a critical residue for pairing with CD94: F107 (CD94), F165 (NKG2A). The corresponding residue is not conserved in NKG2D.
The model also identified that the N- and C-terminals were close together. In the model, shown in
References to heavy chain constant region position numbers here specifically indicate the position of the wild-type constant region sequence starting from the beginning (N-terminus) of CH1 (similar to Uniprot-id:IGHG1_HUMAN (SEQ ID NO:20). For constant light chain positions, numbering is according to Uniprot-id:KAC_HUMAN (SEQ ID NO:10). The amino acids responsible for the ionic interactions in human IgG1 s were identified using an analysis of X-ray structures available for the CH3-CH3 domain-domain interactions of both the GM and KM allotypes, and X-ray structures available for CH1-CKappa and CH1-CLambda interactions.
Specifically, the following KM X-ray structures were analysed: 1HZH, 1ZA6, 1OQX, 1OQO, 1L6X; the following GM X-ray structures were analysed: 1T89, 1T83, 1IIX, 1H3X; the following CH 1-Ckappa X-ray structures were analysed: 1TZG, 1HZH; and the following CH1-Clambda X-ray structure was analysed: 2RCS.
Alignment of the constant domains of GM (SEQ ID NO:8) and KM (SEQ ID NO:9) allotypes are shown in
Using standard methods in available molecular modelling packages, e.g., MOE (Molecular Operating Environment) software available from Chemical Computing Group (www.chemcomp.com), intramolecular ionic interactions were identified. This analysis specifically led to the to the identification of 6 CH3-CH3 GM ionic interactions, 6 CH3-CH3 KM ionic interactions, 2 CH1-CKappa and 2 CH1-CLambda interactions all listed below.
Amino acid residues involved in the above-described interactions were subjected to substitutions in two light-chain heavy chain pairs from different antibodies having different specificities in order to increase the energy of (required for) homodimeric interactions and thereby favor heterodimeric interactions. The same principle can be applied for heavy-light chain interactions, and for soluble heterodimeric receptors.
CH3-Unmodified
<->
CH3-Unmodified
D239
<->
K322
E240
<->
K253
K292
<->
D282
K322
<->
D239
K253
<->
E240
D282
<->
K292.
Suggesting the modifications K322D, K253E, D282K in chain A and D239K, E240K, K292D in chain B leads to a CH3-Modified-A<->CH3-Modified-B interaction with only matching pairs as follows:
D239
<->
K322
E240
<->
K253
K292
<->
D282
D322
<->
K239
E253
<->
K240
K282
<->
D292.
The CH3-Modified-A<->CH3-Modified-A interaction becomes:
D239
<->
D322
E240
<->
E253
K292
<->
K282
D322
<->
D239
E253
<->
E240
K282
<->
K292,
with only charge repulsion pairs.
A similar approach can be applied for the GM, and Heavy light-chain interactions.
Based on the high homology of immunoglobulins, a structural homology can be predicted, and the interactions described above have counterparts for other human isotypes (IgG24) and mouse and rat IgGs. To identify the corresponding residues, an alignment has been performed and is shown in
With respect to conservation of heavy chain, the following can be concluded:
With respect to conservation of light chain, the following can be concluded:
Human CD94 (hCD94) and human NKG2A (hNKG2A) were cloned from a human NK cDNA library purchased from Spring Bioscience (#PHD-138). Primers were designed using sequences from the NCBI database with accession numbers U30610 (hCD94) and AF461812 (hNKG2A). Primers used for hCD94 were
(forward; SEQ ID NO: 40)
5′ GGATCCTCTTTTACTAAACTGAGTATTGAGCCAGC,
and
(reverse, SEQ ID NO: 41)
5′ GCGGCCGCAGATCTATTAAATGAGCTGTTGCTTACAGATATAACG.
Primers used for hNKG2A were
(forward, SEQ ID NO: 42)
5′GGATCCCAGAGGCACAACAATTCTTCCCTG
(reverse, SEQ ID NO: 43)
5′GCGGCCGCAGATCTATTAAAGCTTATGCTTACAATG.
The forward primers contained a BamHI restriction site and the reverse primers contained NotI and BgIII restriction sites. The products were amplified using a touch-down PCR-procedure.
Soluble hCD94 resulted in a 438 bp fragment that was cloned into pCR4blunt-Topo and sequence verified. Soluble hNKG2A (405 bp) was cloned into pCR2.1-TOPO and correct clones were identified by sequencing.
Murine Fc (mFc) was cloned from an IMAGE clone #2651217. Primers used were
(forward, SEQ ID NO: 44)
5′GCTAGCATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCC
CTGGCTATGGATGTGCCCAGGGATTGTGGTTG
(reverse, SEQ ID NO: 45)
5′GGATCCTTTACCAGGAGAGTGGGAGAGGC
The forward primer contains a NheI restriction site followed by CD33 signal peptide and a met and a asp residue. The reverse primer contains a BamHI restriction site. PCR was carried out in 50 μl reaction using 300 ng template, 200 μM dNTP mix, Easy-A High-Fidelity PCR cloning enzyme and buffer from Stratagene #600400. A single denaturering step at 94 C/2 min was followed by 15 cycles as given: 94° C./30 sec; 68° C./30 sec; 72° C./90 sec, ending with 72° C./10 min. The PCR product was separated on a 1% agarose containing EtBr, purified with GFX kit from Amersham Biosciences #27-9602-01 and cloned into pCR4-TOPO (Invitrogen) and sequenced.
mFc was inserted into pcDNA3.1 or pcDNA3.1/Hygro (Invitrogen) using the restriction sites NheI and BamHI. hCD94 was inserted into pcDNA3.1 with mFc using BamHI and NotI (pBF5) and hNKG2A was inserted into pcDNA3.1/Hygro the same way (pBF6). See
pBF6 Encodes the Following (SEQ ID NO:31):
1
MPLLLLLPLLWAGALAMDVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
51
ITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRS
101
VSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIP
151
PPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDG
201
SYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGSQRH
251
NNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKN
301
SSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDS
351
DNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL
pBF5 Encodes the Following (SEQ ID NO:32):
1
MPLLLLLPLLWAGALAMDVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
51
ITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRS
101
VSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIP
151
PPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDG
201
SYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGSSFT
251
KLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESR
301
HLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSAL
351
SQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI
Mutations in the mFc part were achieved using QuikChange mutagenesis on the constructs in Example 3. The single mutation (made as T243Y, Y284T in SEQ ID NO: 21 [IGHG1_MOUSE; IgG1], corresponding to T249Y, Y290T in SEQ ID NO: 14 [GCM_MOUSE; IgG2A]) was carried out using QuikChange II Site-directed Mutagenesis Kit and manual from Stratagene #200523 and the following 2 primer sets:
(SEQ ID NO: 46)
5′GGCCAAGGATAAAGTCAGTCTGTACTGCATGATAACAGACTTC
(SEQ ID NO: 47)
5′GAAGTCTGTTATCATGCAGTACAGACTGACTTTATCCTTGGCC
and
(SEQ ID NO: 48)
5′CAGATGGCTCTTACTTCGTCACCAGCAAGCTCAATGTGCAGAAG
(SEQ ID NO: 49)
5′CTTCTGCACATTGAGCTTGCTGGTGACGAAGTAAGAGCCATCTG.
A single denaturering step at 95° C./30 sec was followed by 15 cycles as given: 95° C./30 sec; 55° C./1 min; 68° C./7 min., ending with 72° C./10 min.
The double mutations (E239K and K292D in one Fc-peptide, D282K and K332E in the other Fc-peptide) were carried out using QuickChange multi Site-directed mutagenesis kit and manual (Stratagene) #200513 and the following primer sets:
E239K:
(SEQ ID NO: 50)
CCATTCCACCTCCCAAGAAGCAGATGGCCAAGG
K292D:
(SEQ ID NO: 51)
GCTCTTACTTCGTCTACAGCGACCTCAATGTGCAGAAGAGCAAC,
and
D282K:
(SEQ ID NO: 52)
GAACACTCAGCCCATCATGAAGACAGATGGCTCTTACTTCG
K322E:
(SEQ ID NO: 53)
CCACCATACTGAGGAGAGCCTCTCCCACTCTCCTGG.
A single denaturering step at 95° C./1 min was followed by 30 cycles as given: 95° C./1 min; 55° C./1 min; 65° C./13.5 min.
NKG2A-2×GGS-CD94 was cloned into pcDNA3.1/hygro with mFc in the BamHI/Notl site (pBF17). See
pBF17 Encodes the Following (SEQ ID NO:39):
1
MPLLLLLPLLWAGALAMDVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT
51
ITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRS
101
VSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIP
151
PPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDG
201
SYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKGSQRH
251
NNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKN
301
SSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDS
351
DNAELNCAVLQVNRLKSAQCGSSIIYHCKHKLGGSGGSRSSFTKLSIEPA
401
FTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNESRHLCASQK
451
SSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPS
501
FETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI
All the constructs in Examples 2-5 were expressed transient in HEK293 cells using 293fectin #12347-019 from Invitrogen for transfection. 5×105 cells/ml were seeded in a 125 ml Erlen-Meyer flask. The following day, the cells were transfected as follows: 30 μg DNA was diluted in 1 ml Opti-Mem and 40 μl 293 fectin was diluted in 960 μl Opti-Mem. After 5 min. at room temperature (RT) the two mixtures were mixed and incubated 25 min. at RT before the addition to the cell culture. Media was harvested 4-6 days later by centrifugation at 1500×g for 15 min. The results were visualized by western blot analysis, where all the blots were probed with goat-anti-mouse-IgG.
Exemplary results of western blot analysis are shown in
Six different variants of CD94-mFc and NKG2A-mFc fusion proteins were purified from cell culture supernatants by Protein A affinity chromatography. The fusion proteins were CD94-mFc homodimer, CD94-2×GGS-NKG2A-mFc, NKG2A-mFc, CD94-mFc(T249Y) NKG2A-mFc(Y290T), CD94-mFc NKG2A-mFc, CD94-mFc(E239K, K292D) NKG2A-mFc(D282K, K332E). CD94/NKG2A-mouse Fc fusion proteins were produced using serum-free medium. CD94/NKG2A-mouse Fc fusion protein was expressed from CHO-DUKX B11 cells.
Purification of CD94/NKG2A-mouse Fc fusion proteins was performed as follows. Cell culture supernatants were sterile filtered and applied to a 10 ml Protein A sepharose column (Sigma, St. Louis, Mo.) packed in a XK16 column (GE-Healthcare, Hillerod, Denmark) using an ÄKTA explorer FPLC system (GE-Healthcare, Hillerod, Denmark). The protein A column was equilibrated in 20 mM Tris pH 7.5 at 6 ml/min. After sample application, the column was washed until UV absorbance at 280 nm was low. The bound proteins were eluted using a step gradient from 0 to 100% 50 mM Glycine pH 2.7 and 2 ml fractions were collected. Fractions containing the proteins of interest were pooled and titrated to neutral pH. In some cases the pooled proteins were concentrated using a Vivaspin 20, 30.000 MWCO (Sartorius, Roskilde, Denmark) and buffer exchanged into PBS buffer. Protein concentrations were determined by OD280 with an extension coefficient of (1.5 g/l)−1×cm−1.
The purified proteins were analyzed by 1-D SDS-PAGE on 4-12% NuPage gels (In-Vitrogen, Carlsbad, Calif.) running in MOPS buffer. 1-D gels were stained using Gelcode coomassie stain (Pierce, Rockford, Ill.) or blotted onto PVDF membranes (InVitrogen, Carlsbad, Calif.).
The antibodies Z199 (anti-NKG2A, Beckman Coulter, Fullerton, Calif.) and HP-3D9 (anti-CD59, Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) were prepared for western-blotting by labeling with Cy3 and Cy5 using Cy-dye protein labeling kits (GE-Healthcare, Hillerod, Denmark). After blotting, the membranes were blocked in 5% blotto (Pierce, Rockford, Ill.) and incubated with both antibodies diluted 1:200 in washing buffer. After washing the blots were scanned on either an Ettan Dige Imager or a Typhoon Trio+scanner (GE-Healthcare, Hillerod, Denmark) using the settings for the two different fluorophors.
Results Proteins purified from 330-350 ml serum-free culture supernatant from HEK293 cells resulted in 0.08 to 1.17 mg protein. See Table 3. The largest protein amounts were purified from cells expressing the two plasmids with a single mutation in murine immunoglobulin constant region. Analysis of the purified proteins by 1-D SDS PAGE showed that the purification yielded quite pure proteins with no obvious contaminants present. The main band corresponded to dimer formation of the proteins, whereas a higher molecular weight species corresponded to tetramer formation.
TABLE 3
Proteins purified from serum free expression in HEK293 freestyle cells
Protein
Plasmid Constructs
Amount (mg)
CD94-mFc homodimer
pBF5
0.37
CD94-2xGGS-NKG2A-mFc
pBF17
0.18
NKG2A-mFc
pBF6
0.27
CD94-mFc T249Y
pBF19 and pBF20
1.7
NKG2A-mFc Y290T
CD94-mFc
pBF5 and pBF6
0.88
NKG2A-mFc
CD94-mFc E239K, K292D
pBF21 and pBF22
0.08
NKG2A-mFc D282K, K332E
To ascertain that the purified proteins were combinations of both CD94 and NKG2A, a western blot were made, where the two antibodies Z199 and HP-3D9 (against NKG2A and CD94 respectively) were used. The antibodies were labeled with fluorescence dyes, making it possible to use both antibodies on a single blot (
A stable CHO cell line (CHO-DUKX B11) expressing the single-chain CD94-2×GGS-NKG2A-mFc constructs was used to produce protein for Biacore analysis. 215 ml cell culture supernatant was purified on Protein A column and buffer exchanged into PBS. The resulting protein was analyzed by 1-D SDS PAGE and western blotting using the two antibodies Z199 and HP-3D9 labeled with fluorescence dyes (
NKG2A was subcloned BamHI-BgIII in BamHI+dephosphorylated (using calf intestine phophatase) in Trx-hexaHis pET32a to produce pISNN415, encoding the following sequence (SEQ ID NO:54) (
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLN
60
IDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHH
120
HHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSQRHNNSSLNTRTQKA
180
RHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSS
240
WIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL
300
**
302
The human NKG2A (residues 99-233 of SEQ ID NO:1) extracellular domain was expressed in E. coli. NKG2A was fused N-terminally to Thioredoxin (Trx) and hexaHis tag. The expected molecular weight was 33 kDa. Trx and hexaHis can be proteolytically removed using Enterokinase. Trx-NKG2A was expressed at approximately 10 mg/L in 500 mL lab-scale cultures (
CD94 was subcloned BamHI-BgIII in BamHI+dephosphorylated (using calf intestine phophatase) hexaHis-GST XPNNB pET15b to produce pISNN427, encoding the following sequence (SEQ ID NO:55) (
MGSSHHHHHHSSGLVPRGSHMLESPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEG
60
DKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGA
120
VLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDA
180
LDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK
240
SDLVPRGSSFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNES
300
RHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFE
360
TFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI**
396
The human CD94 (residues 34-179 of SEQ ID NO:2) extracellular domain was expressed in E. coli. CD94 was fused N-terminally to hexaHis and Glutathione-S-transferase (GST). The expected molecular weight was 46 kDa. hexaHis-GST can be removed by cleavage with Thrombin. The expression level of GST-CD94 was approximately 50 mg/L in 500 mL lab-scale cultures. Expression was obtained at 30° C. The fusion proteins were expressed in an insoluble form. Various lysis methods were screened in this particular experiment (freeze-thaw (fz) cycles using TEN (tris, EDTA, NaCl), or detergent based lysis using BugBuster (Novagen) or PopCulture (Novagen)) (
Primers used were:
2 × GGS - up:
(SEQ ID NO: 56)
AGCTTGGCGGTAGCGGCGGTAGCA
2 × GGS dwn:
(SEQ ID NO: 57)
GATCTGCTACCGCCGCTACCGCCA
2 × GGS insertion dwn:
(SEQ ID NO: 58)
GTTGTGCCTCTGGCTACCGCCGCTACCGCCAATGAG-CTGTTGC
NKG2A-2×GGS-CD94. First, the NKG2A 3′ end was altered in pBluescript-SK(−). The annealed oligos (‘2×GGS-up’ and ‘2×GGS dwn’) which at the same time added a hydrophilic linker and removed stopcodons of the original clone was cloned HindIII-BgIII in NKG2A-pBluescript-SK(−) (pISNN425) (
NKG2A-2×GGS-CD94 (NKG2A-2×GGS-CD94-pBluescript-SK(−))) was subcloned BamHI-BgIII in BamHI restricted and +de-phosphorylated (using calf intestine phosphatase)−hexaHis-GST XPNNB pET15b or BamHI-BgIII in BamHI+dephosphorylated Trx-hexaHis pET32a (
CD94-2×GGS-NKG2A. In another approach, CD94 is inserted BamHI-BgIII in BamHI restricted and dephosphorylated NKG2A pBluescript-SK(−) (pISNN425). A linker (2×GGS insertion dwn) which at the same time removes a stop codon and adds a hydrophilic linker is inserted by site-directed mutagenesis. The primer is 5-Phosphorylated and the ‘Quick-change multi site-directed mutagenesis kit’ (purchased form Stratagene) is used. T7 and T3 primers are used for sequence verification. CD94-2×GGS-NKG2A heterodimers are subcloned BamHI-BgIII in BamHI+dephosphorylated hexaHisGST XPNNB pET15b og BamHI-BgIII in BamHI+dephosphorylated Trx-hexaHis pET32a. See
The constructs are expressed in BL21 (DE3).
The amino-acid sequence for hexaHis-GST NKG2A-2×GGS-CD94 is (SEQ ID NO:37):
MGSSHHHHHHSSGLVPRGSHMLESPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEG
60
DKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGA
120
VLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDA
180
LDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK
240
SDLVPRGSQRHNNSSLNTRTQKARHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSK
300
NSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAV
360
LQVNRLKSAQCGSSIIYHCKHKLGGSGGSRSSFTKLSIEPAFTPGPNIELQKDSDCCSCQ
420
EKWVGYRCNCYFISSEQKTWNESRHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYS
480
EEHTAWLWENGSALSQYLFPSFETFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI**
539
The amino-acid sequence for Trx-hexaHis-NKG2A-2×GGS-CD94 is (SEQ ID NO:38):
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLN
60
IDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHH
120
HHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIGSQRHNNSSLNTRTQKA
180
RHCGHCPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSS
240
WIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL
300
GGSGGSRSSFTKLSIEPAFTPGPNIELQKDSDCCSCQEKWVGYRCNCYFISSEQKTWNES
360
RHLCASQKSSLLQLQNTDELDFMSSSQQFYWIGLSYSEEHTAWLWENGSALSQYLFPSFE
420
TFNTKNCIAYNPNGNALDESCEDKNRYICKQQLI**
456
Human NKG2A (residues 99-233 of SEQ ID NO:1) and human CD94 (residues 34-179 of SEQ ID NO:2) extracellular parts, separated by a gly-gly-ser-gly-gly-ser linker, were expressed as a single-chain fusion in the E. coli strain BL21 (DE3). Two different NKG2A-CD94 fusion constructs were expressed carrying respectively Thioredoxin (Trx) and hexaHis tag or hexaHis and Glutathione-S-transferase (GST) at their N-termini. Trx-hexaHis-NKG2A-2×GGS-CD94 was expressed at approximately 100 mg/L in 500 mL lab-scale cultures. The expression level of hexaHis-GST-NKG2A-2×GGS-CD94 was approximately 70 mg/L in 500 mL lab-scale cultures. Trx-hexaHis may be proteolytically removed using Enterokinase. hexaHis-GST can be removed by cleavage with Thrombin. Expression was obtained at 37° C. The fusion proteins were highly expressed in the insoluble pellet fractions (p) (
Isolated cells from 250 ml fermentation were washed once with 50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA. The obtained pellet was resuspended in 50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA, 10 w/v % sucrose, 5 mM DTT, 5 mM benzamidine, and lysed in a French press. The insoluble inclusion bodies were collected by centrifugation, washed and dissolved in 6M guadiniumhydrochlorid, 100 mM Tris pH 8, 40 mM DTT.
The solubilized protein was diluted into 20 ml refolding buffer 50 mM Tris pH 8.2, 750 mM arginine, 10 mM NaCl, 0.5 mM KCl, 0.5 g/L PEG3350, 2 mM MgCl2, 2 mM CaCl2, 4 mM cystine, 1.5 mM DTT. The refolding mixture was left at 5° C. over night.
The refolded protein was diluted 5 times with 10 mM Tris pH 8 and purified by application to a 5 ml Q sepharose fast flow column. After wash with 2 Cv 10 mM Tris pH 8, 50 mM NaCl, the protein was eluted by a linear gradient over 10 CV to 10 mM Tris pH 8, 2 M NaCl.
The isolated cells from 1 L fermentation were washed once with 50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA. The obtained pellet was resuspended in 50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA, 10 w/v % sucrose, 5 mM DTT, 5 mM benzamidine, and lysed in a French press. The insoluble inclusion bodies were collected by centrifugation, washed and dissolved in 6M guadiniumhydrochlorid, 100 mM Tris pH 8, 40 mM DTT.
The solubilized protein was diluted into 100 ml refolding buffer 50 mM Tris pH 8.2, 750 mM aginine, 10 mM NaCl, 0.5 mM KCl, 0.5 g/L PEG3350, 2 mM MgCl2, 2mM CaCl2, 4 mM cystine, 1.5 mM DTT. The refolding mixture was left at 5 ° C. over night.
The refolded protein was diluted 5 times with 10 mM Tris pH 8 and purified by application to a 5 ml Q sepharose fast flow column. After wash with 2 Cv 10 mM Tris ph 8, 50 mM NaCl, the protein was eluted by a linear gradient over 10 CV to 10 mM Tris pH 8, 2 M NaCl.
Isolated cells from 1 L fermentation are washed once with 50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA. The obtained pellet is resuspended in 50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA, 10 w/v % sucrose, 5 mM DTT, 5 mM benzamidine, and lysed in a French press. The insoluble inclusion bodies are collected by centrifugation, washed and dissolved in 6M guadiniumhydrochlorid, 100 mM Tris pH 8, 40 mM DTT.
The solubilized protein is diluted into 20 ml refolding buffer 50 mM Tris pH 8.2, 750 mM aginine, 10 mM NaCl, 0.5 mM KCl, 0.5 g/L PEG3350, 2 mM MgCl2, 2 mM CaCl2, 4 mM cystine, 1.5 mM DTT. The refolding mixture is left at 5° C. over night.
The refolded protein is diluted 5 times with 10 mM Tris pH 8 and is purified by application to a 5 ml Q sepharose fast flow column. After wash with 2 Cv 10 mM Tris ph 8, 50 mM NaCl, the protein is eluted by a linear gradient over 10 CV to 10 mM Tris pH 8, 2 M NaCl.
The following constructs were analyzed for binding to the immobilized antibodies HP-3D9, Z199 and rabbit anti-mouse (RaM), with subsequent binding of HLA-E tetramer:
CD94-mFc homo dimer
CD94-NKG2A-mFc singlechain
NKG2A-mFc homo dimer
CD94-mFc/NKG2A heterodimer with single mutation
CD94-mFc/NKG2A heterodimer
CD94-mFc/NKG2A heterodimer with double mutation.
Materials
Surface plasmon resonance studies were performed on a Biacore3000 instrument (Biacore AB, Uppsala, Sweden). Immobilization of ligands were conducted on a CM5 sensor chip (Biacore AB), using a standard amine coupling kit as described by the manufacture (Biacore AB).
HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Polysorbat 20 (v/v)) was used as running buffer, and for all dilutions. Regeneration of the sensor chip was performed by a short pulse (15 ul, Flow 30 ul/min) of 10 mM glycin-HCl pH 1.8.
All experiments were performed at flow rate 10 ul/min. at 25° C. Data was analyzed using Biaevaluation 4.1 software.
Anti-CD94 monoclonal antibody HP-3D9 (BD Pharmingen, cat #555887), anti-CD94/NKG2A monoclonal antibody Z199 (Immunotech, France) and Rabbit anti-mouse (RaM) polyclonal antibody (Biacore AB), were immobilized by amine coupling to a level of ˜5000 RU in individual flow cells on a CM5 sensor chip.
Binding of CD94INKG2A Constructs to Anti-CD94 and Anti-CD94/NKG2A Monoclonal Antibodies, and Competition on HLA-E Tetramer Binding
All constructs were diluted to 1 ug/ml in HBS-EP. HLA-E tetramer was tested in 5 ug/ml conc. Each cycle consisted of initial injection of a given construct, followed by injection of HLA-E tetramer. Construct and HLA-E tetramer were injected for 3 min., each followed by a 2.5 min. dissociation phase, prior to regeneration of the sensor chip surface.
All constructs were tested as stored at 4° C., and at −20° C.
All constructs, except NKG2A-mFc homodimer, demonstrated binding to immobilized HP-3D9. Binding of the individual constructs to HP-3D9 appeared not to be affected by the storage conditions, except for the CD94-mFc/NKG2A heterodimer with single mutation construct, where an apparent reduction in the on-rate could be detected for the construct stored at −20° C. However the construct did bind immobilized HP-3D9.
The apparent off-rate of the CD94-mFc/NKG2A heterodimer with double mutation construct was higher as compared to the other constructs which demonstrated apparent identical dissociation patterns. All constructs, except the CD94-mFc/NKG2A heterodimer with double mutation, demonstrated stable binding during the 2.5 min. dissociation phase.
No binding of HLA-E tetramer to any of the individual constructs in complex with HP-3D9 was detected.
Binding to immobilized Z199 was observed for the CD94-NKG2A-mFc singlechain, CD94-mFc/NKG2A heterodimer and CD94-mFc/NKG2A heterodimer with double mutation constructs irrespective of the storage conditions. Also, binding of the CD94-mFc/NKG2A heterodimer with single mutation construct stored at 4° C. was detected, while the same construct stored at −20° C. demonstrated no binding. Although binding of the CD94-mFc/NKG2A heterodimer and CD94-mFc/NKG2A heterodimer with single mutation constructs was detected, their association to immobilized Z199 appeared reduced as compared to that of the CD94-NKG2A-mFc singlechain and CD94-mFc/NKG2A heterodimer with double mutation constructs.
While the binding of the CD94-NKG2A-mFc singlechain, CD94-mFc/NKG2A heterodimer and CD94-mFc/NKG2A heterodimer with single mutation (4° C. storage) appear stable during the 2.5 min. dissociation phase, dissociation of CD94-mFc/NKG2A heterodimer with double mutation was fairly fast.
Binding of HLA-E tetramer to a construct in complex with Z199, was detected for the CD94-NKG2A-mFc singlechain construct. The binding of HLA-E tetramer appeared stable during the 2.5 min. dissociation period.
All constructs, except NKG2A-mFc homo dimer, bind to immobilized RaM. Again, the association of the CD94-mFc/NKG2A heterodimer with single mutation construct stored at −20° C. appear reduced compared to the same construct stored at 4° C.
Binding of HLA-E tetramer to complexes between RaM and the individual constructs was observed for all constructs, except for the NKG2A-mFc homodimer. The relative highest amounts of HLA-E tetramer bound, was detected for the CD94-NKG2A-mFc singlechain and CD94-mFc/NKG2A heterodimer with double mutation complexes.
E Coli expressed THX-His-tag-NKG2a-GSS-GSS-CD94-His-Tag/pET32a was analyzed for binding to immobilized HP-3D9 and Z199 monoclonal antibodies (see Example 18). Two different preparations prepared at pH 6.0, 7.0 and 8.0 respectively, were tested; one obtained from a 250 ml fermentation (B3), and one from a 1 L fermentation (B12) (see Examples 15 and 16).
The preparations from the 250 ml fermentation were tested in 1:10 dilution in HBS-EP, and injected for 3 min., followed by 2.5 min. dissociation. The preparations bound to both immobilized HP-3D9 and immobilized Z199, with higher amounts bound to immobilized HP-3D9 (
The preparations from the 1 L fermentation were diluted to 10 ug/ml, and injected for 3 min., followed by 2.5 min. dissociation. This preparation also demonstrated binding to both immobilized HP-3D9 and Z199 (
Soluble human NKG2C was ordered from Geneart, Regensburg, Deutschland. The sequence was ordered with a 5′ BamHI restriction site and a 3′ HindIII restriction site in order to easily replace hNKG2A from pBF17 resulting in pBF74 (NKG2C/CD94-Fc) (SEQ ID NO:59):
1 MPLLLLLPLL WAGALAMDVP RDCGCKPCIC TVPEVSSVFI FPPKPKDVLT
51 ITLTPKVTCV VVDISKDDPE VQFSWFVDDV EVHTAQTKPR EEQFNSTFRS
101 VSELPIMHQD WLNGKEFKCR VNSAAFPAPI EKTISKTKGR PKAPQVYTIP
151 PPKEQMAKDK VSLTCMITDF FPEDITVEWQ WNGQPAENYK NTQPIMDTDG
201 SYFVYSKLNV QKSNWEAGNT FTCSVLHEGL HNHHTEKSLS HSPGKGSFLE
251 QNNSSPNTRT QKARHCGHCP EEWITYSNSC YYIGKERRTW EESLLACTSK
301 NSSLLSIDNE EEMKFLASIL PSSWIGVFRN SSHHPWVTIN GLAFKHKIKD
351 SDNAELNCAV LQVNRLKSAQ CGSSMIYHCK HKLGGSGGSR SSFTKLSIEP
401 AFTPGPNIEL QKDSDCCSCQ EKWVGYRCNC YFISSEQKTW NESRHLCASQ
451 KSSLLQLQNT DELDFMSSSQ QFYWIGLSYS EEHTAWLWEN GSALSQYLFP
501 SFETFNTKNC IAYNPNGNAL DESCEDKNRY ICKQQLI
The construct comprise the signal peptide CD33 (SEQ ID NO:30), an 227 Fc-portion (Hinge-CH2-CH3) (SEQ ID NO:21), linker (GS), residues 96-231 of hNKG2C (SEQ ID NO:3), an GGSGGSRSS spacer and residues 34 to 180 of hCD94 (SEQ ID NO:2).
Mutated NKG2A-Portion Constructs for Mammalian Cell Expression
The amino acid sequences of the soluble portions of NKG2A and NKG2C used in the single chain constructs differ only in residues 99, 100, 101, 106, 167, 168, 170, 189 and 197, using the numbering of the alignment in
A series of CD94/NKG2C-single chain constructs were developed based on the single chain construct (Sig)-(mFc)-GS-(NKG2A)-GGS-GGS-RSS-(CD94) (SEQ ID NO:39). This construct comprises residues 34 to 180 of hCD94 (SEQ ID NO:2), residues 99-233 of hNKG2A (SEQ ID NO:1), an GGSGGSRSS spacer between the CD94 and NKG2A portions, and an 227 Fc-portion. The series was prepared with iterative mutations listed in Table 4 below:
TABLE 4
Iterative mutations to convert ssCD94/NKG2A to ssCD94/NKG2C
construct
Mut1
Q99L, R100E, H101Q, L106P
Mut2
S167A, I168S, S170L
Mut3
M189I
Mut4
E197K
Mut5
I225M
Mutations in the hNKG2A part were achieved using quikchange mutagenesis on the construct pBF17(SEQ ID NO:39). The primers used in the quikchange mutagenesis were:
Mut1 LEQ-P; Forward (SEQ ID NO:60), Reverse (SEQ ID NO:61)
Mut2 AS-L; Forward (SEQ ID NO:62), Reverse (SEQ ID NO:63)
Mut3 I; Forward (SEQ ID NO:64), Reverse (SEQ ID NO:65)
Mut4 K; Forward (SEQ ID NO:66), Reverse (SEQ ID NO:67)
Mut5 M; Forward (SEQ ID NO:68); Reverse (SEQ ID NO:69)
These mutations were carried out using QuikChange II Site-directed Mutagenesis Kit and manual from Stratagene #200523: A single denaturering step at 95° C./30 sec was followed by 15 cycles as given: 95° C./30 sec; 55° C./1 min; 68° C./7 min., ending with 72° C./10 min.
Materials & Methods
Surface plasmon resonance (SPR) measurements were performed on a Biacore T100 apparatus (Biacore GE Healthcare) at 25° C. In all Biacore experiments HBS-EP+buffer (Biacore GE Healthcare) served as running buffer and sensorgrams were analyzed with Biaevaluation 4.1 and Biacore T100 Evaluation softwares. The antibodies used were anti-NKG2A (humZ270 and Z199), anti-NKG2C (Immunotech-Beckman Coulter), and anti-NKG2D (ON72).
Protein Immobilization.
Recombinant proteins were immobilized covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5 (chip). The chip surface was activated with EDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Proteins were diluted to 10 μg/ml in coupling buffer (10 mM acetate, pH 5.6) and injected until the appropriate immobilization level was reached (i.e. 700, 630 and 400 RU for 2A, 2C and 2D respectively).
Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).
Binding Study
For simple binding experiments, antibodies were injected at a constant concentration of 10 μl g/ml for 1 minute at a flow rate of 10 μl/min onto the scCD94/NKG2A, scCD94/NKG2C and NKG2D-Fc recombinant protein chips. After each cycle, the chips were regenerated by an eight second injection of 500 mM NaCl and 10 mM NaOH buffer at flow rate of 40 μl/min. For each antibody the sensorgrams obtained on the different protein chips were superimposed and normalised to an arbitrary value of 100 RU for the Y axis.
Affinity Measurement
For kinetic experiments, serial dilutions from 0.078 to 5 nanoM of soluble antibodies were injected for 2 min at a constant flow rate of 40 μl/min on dextran layers containing immobilized scCD94/NKG2A recombinant proteins and allowed to dissociate for 3 min before regeneration by an eight second injection of 500 mM NaCl and 10 mM NaOH buffer.
The resulting sensorgrams were analysed by global fitting using the appropriate Langmuir model.
HLA-E Tetramers Binding Inhibition
For inhibition experiments, antibodies were injected at a constant concentration of 10 μg/ml on dextran layers containing immobilized scCD94/NKG2A recombinant target proteins. Each competition cycle consisted of three 2 min injection steps at a constant flow rate of 10 μl/min. Firstly, the antibody is injected twice. Secondly, without removing the bound antibody, the HLA-E tetramer at 8 μg/ml is injected and sensorgrams and RU values are monitored. The binding signals of the HLA-E tetramer in the presence of antibodies are compared to those obtained when the HLA-E tetramer is injected directly on nude recombinant scCD94/NKG2A proteins. The percentage of inhibition (1%) was determined from RU values obtained 10 second after the end of injections, using the following formula: 1% =(1−(RU+Ab/RUNone))*100. RU+Ab and RUNone are HLA-E binding RU values monitored respectively in the presence and in the absence of an antibody. The sensorgrams corresponding to HLA-E injection (third step) were aligned to zero at the injection start for both X and Y axis and superimposed.
After each cycle, the chips were regenerated by an eight second injection of 500 mM NaCl and 10 mM NaOH buffer at flow rate of 40 μl/min.
Results
The binding of humZ270 and humZ199 antibodies to recombinant NKG2A, NKG2C and NKG2D proteins was analysed by SPR (
The scCD94/NKG2C chip was checked using anti-NKG2C-PE (FAB138P, R&D Systems), injected at 2.5 μg/ml for 1 min onto the NKG2C chip. This antibody binds well to native NKG2C expressed at the cell surface. The antibody bound to the scCD94/NKG2C chip, indicating the presence of immobilized proteins in a proper status that mimics the cell surface situation. The NKG2D-Fc chip was checked using humON72 antibodies.
To analyse the HLA-E binding properties, the binding of HLA-E tetramers to scCD94/NKG2A chips was monitored in the absence and in the presence of humZ270. The saturation of scCD94/NKG2A chips using humZ270 antibodies completely inhibits the binding of HLA-E tetramers (IZ270=81.8%).
All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.
All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way,
Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
The terms “a” and “an” and “the” and similar referents as used in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by “about,” where appropriate).
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents,
The description herein of any aspect or embodiment of the invention using terms such as “comprising”, “having”, “including” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that “consists of”, “consists essentially of”, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).
This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.
Padkær, Søren Berg, Spee, Petrus Johannes Louis, Nørby, Inga Sig Nielsen, Friedrichsen, Birgitte Nissen
| Patent | Priority | Assignee | Title |
| 11718683, | Mar 10 2016 | Aperisys, Inc.; The General Hospital Corporation | Antigen-binding fusion proteins with modified HSP70 domains |
| 11896616, | Mar 27 2017 | National University of Singapore | Stimulatory cell lines for ex vivo expansion and activation of natural killer cells |
| Patent | Priority | Assignee | Title |
| 6018026, | Jan 22 1988 | ZymoGenetics, Inc. | Biologically active dimerized and multimerized polypeptide fusions |
| 6238890, | Feb 18 1994 | Washington University | Single chain forms of the glycoprotein hormone quartet |
| 6262244, | Mar 28 1991 | Regents of the University of Minnesota; Novartis AG | DNA and amino acid sequence specific for natural killer cells |
| 6833441, | Aug 01 2001 | ABMAXIS, INC | Compositions and methods for generating chimeric heteromultimers |
| 20030078385, | |||
| 20030195338, | |||
| 20040072256, | |||
| 20040138417, | |||
| WO158957, | |||
| WO203237, | |||
| WO208272, | |||
| WO2101006, | |||
| WO212345, | |||
| WO222153, | |||
| WO3012069, | |||
| WO9206204, | |||
| WO9533057, | |||
| WO9734631, | |||
| WO9850431, | |||
| WO9928747, | |||
| WO9929732, | |||
| WO9937772, |
| Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
| Jun 22 2007 | Novo Nordisk A/S | (assignment on the face of the patent) | / | |||
| Jan 16 2009 | FRIEDRICHSEN, BIRGITTE NISSEN | Novo Nordisk A S | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 022812 | /0529 | |
| Jan 21 2009 | NORBY, INGA SIG NIELSEN | Novo Nordisk A S | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 022812 | /0529 | |
| May 20 2009 | PADKAER, SOREN BERG | Novo Nordisk A S | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 022812 | /0529 | |
| May 25 2009 | SPEE, PETRUS JOHANNES LOUIS | Novo Nordisk A S | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 022812 | /0529 | |
| Aug 05 2016 | Novo Nordisk A S | INNATE PHARMA S A | ASSIGNMENT OF ASSIGNORS INTEREST SEE DOCUMENT FOR DETAILS | 039431 | /0027 |
| Date | Maintenance Fee Events |
| Sep 17 2014 | M1551: Payment of Maintenance Fee, 4th Year, Large Entity. |
| Oct 26 2018 | M1552: Payment of Maintenance Fee, 8th Year, Large Entity. |
| Dec 12 2022 | REM: Maintenance Fee Reminder Mailed. |
| May 29 2023 | EXP: Patent Expired for Failure to Pay Maintenance Fees. |
| Date | Maintenance Schedule |
| Apr 26 2014 | 4 years fee payment window open |
| Oct 26 2014 | 6 months grace period start (w surcharge) |
| Apr 26 2015 | patent expiry (for year 4) |
| Apr 26 2017 | 2 years to revive unintentionally abandoned end. (for year 4) |
| Apr 26 2018 | 8 years fee payment window open |
| Oct 26 2018 | 6 months grace period start (w surcharge) |
| Apr 26 2019 | patent expiry (for year 8) |
| Apr 26 2021 | 2 years to revive unintentionally abandoned end. (for year 8) |
| Apr 26 2022 | 12 years fee payment window open |
| Oct 26 2022 | 6 months grace period start (w surcharge) |
| Apr 26 2023 | patent expiry (for year 12) |
| Apr 26 2025 | 2 years to revive unintentionally abandoned end. (for year 12) |